Carboxylation mechanism and stereochemistry of crotonyl ... - PNAS

Carboxylation mechanism and stereochemistry of crotonyl-CoA carboxylase/reductase, a carboxylating enoyl-thioester reductase Tobias J. Erba,1, Volker Brechtb, Georg Fuchsa, Michael Mu¨llerb, and Birgit E. Alberc aMikrobiologie, Institut fu ¨ r Biologie II, Scha¨nzlestrasse 1 and bPharmazeutische und Medizinische Chemie, Institut fu¨r Pharmazeutische Wissenschaften, Albertstrasse 25, Albert-Ludwigs-Universita¨t Freiburg, 79104 Freiburg im Breisgau, Germany; and cDepartment of Microbiology, Ohio State University, 484 West 12th Avenue, Columbus, OH 43210

alcohol dehydrogenase 兩 biocatalysis 兩 enoyl reductase

T

he use of enzymes in organic chemistry has been increasing steadily in recent years, because enzymatic catalysis provides some advantages over classical synthesis methods. Besides the capability to promote reactions under mild conditions, the high regio- and stereoselectivity of biocatalysts has received much attention (1). Reductases represent an important class of enzymes that are used in organic synthesis, with alcohol dehydrogenases and enoate reductases as the most prominent examples. Enoate reductases are unique in their ability to reduce selectively CAC bonds in ␣,␤-unsaturated carbonyl compounds and to create thereby up to 2 stereogenic centers in the target molecule. This chemo- and stereoselectivity makes enoate reductases an important addition to the synthetic toolbox (1–4). Another challenge in organic synthesis is the introduction of carboxyl groups into a target molecule (5). Direct carboxylations of organic substrates are poorly represented in organic synthesis, and the few examples, such as the Kolbe–Schmitt reaction or the Grignard reaction require quite harsh conditions (Kolbe–Schmitt) or an inert atmosphere and nonaqueous solvents (Grignard) (6). Although carboxylation reactions occur widely in nature, carboxwww.pnas.org兾cgi兾doi兾10.1073兾pnas.0903939106

ylating enzymes are rarely used in organic synthesis. The only prominent examples reported thus far are phenylphosphate carboxylase (6) and pyrrole-2-carboxylate decarboxylase (7). The application of most carboxylases is limited, because many carboxylating enzymes require either complex or unstable substrates (e.g., ribulose-1,5-bisphosphate, phosphorenolpyruvate), depend on cofactors (e.g., ATP, biotin), metal ions, or are multienzyme complexes difficult to prepare for synthetic purposes [supporting information (SI) Table S1]. We recently reported the discovery of an enzyme, crotonyl-CoA carboxylase/reductase (Ccr) from Rhodobacter sphaeroides, that represents a type of carboxylase and catalyzes the reductive carboxylation of (E)-crotonyl-CoA to ethylmalonyl-CoA with reduced nicotinamide adenine dinucleotide phosphate (NADPH) as reductant (8). Acryloyl-CoA is accepted as an alternative substrate analogue by the enzyme with 40% relative activity (compared with vmax of crotonyl-CoA carboxylation), yielding methylmalonyl-CoA. Interestingly, crotonyl-CoA carboxylase/reductase is related to dehydrogenases that reduce CAC or CAO bonds, and Ccr also catalyzes the reduction of (E)-crotonyl-CoA to butyryl-CoA in the absence of HCO3/CO2, albeit with only 10% of maximal activity (compared with vmax of crotonyl-CoA carboxylation), indicating that the carboxylation reaction is the physiologically relevant reaction (Fig. 1A). The properties of Ccr are summarized in Table 1. In this work, the carboxylation mechanism and the stereochemical course of both the carboxylation reaction and the reduction reaction of Ccr were investigated in detail. The results reported herein led to a reassessment of the stereochemical diversity with respect to the amino acid sequence diversity of enoyl (-thioester) reductases and revealed interesting aspects about the evolution of these enzymes, with possible implications for protein engineering and the use of those enzymes in biocatalysis. Results Mechanism of Carboxylation (Carboxylating Species). To investigate

the mechanism of carboxylation, the oxidation of NADPH in a mixture of crotonyl-CoA* and Ccr was followed spectrophotometrically at 360 nm upon the addition of either CO2 or HCO⫺ 3 . In principle, either CO2 or HCO⫺ 3 , can serve as active species of CO2 in enzymatic carboxylation reactions (9). Because the hydration of ⫹ CO2 (‘‘dissolved’’ CO2 ⫹ H2O D H2CO3 D HCO⫺ 3 ⫹ H ) is slow at temperatures ⬍20 °C, a difference in the initial enzymatic rate can be observed, when nonsaturating concentrations of either dissolved CO2 or HCO⫺ 3 are added to start the reaction at low Author contributions: T.J.E., M.M., and B.E.A. designed research; T.J.E. and V.B. performed research; T.J.E., V.B., and M.M. analyzed data; and T.J.E., G.F., M.M., and B.E.A. wrote the paper. The authors declare no conflict of interest. 1To

whom correspondence should be addressed. E-mail: [email protected].

*From here on, ‘‘crotonyl-CoA’’ is used for (E)-crotonyl-CoA. This article contains supporting information online at www.pnas.org/cgi/content/full/ 0903939106/DCSupplemental.

PNAS 兩 June 2, 2009 兩 vol. 106 兩 no. 22 兩 8871– 8876

CHEMISTRY

Chemo- and stereoselective reductions are important reactions in chemistry and biology, and reductases from biological sources are increasingly applied in organic synthesis. In contrast, carboxylases are used only sporadically. We recently described crotonyl-CoA carboxylase/reductase, which catalyzes the reduction of (E)crotonyl-CoA to butyryl-CoA but also the reductive carboxylation of (E)-crotonyl-CoA to ethylmalonyl-CoA. In this study, the complete stereochemical course of both reactions was investigated in detail. The pro-(4R) hydrogen of NADPH is transferred in both reactions to the re face of the C3 position of crotonyl-CoA. In the course of the carboxylation reaction, carbon dioxide is incorporated in anti fashion at the C2 atom of crotonyl-CoA. For the reduction reaction that yields butyryl-CoA, a solvent proton is added in anti fashion instead of the CO2. Amino acid sequence analysis showed that crotonyl-CoA carboxylase/reductase is a member of the medium-chain dehydrogenase/reductase superfamily and shares the same phylogenetic origin. The stereospecificity of the hydride transfer from NAD(P)H within this superfamily is highly conserved, although the substrates and reduction reactions catalyzed by its individual representatives differ quite considerably. Our findings led to a reassessment of the stereospecificity of enoyl(-thioester) reductases and related enzymes with respect to their amino acid sequence, revealing a general pattern of stereospecificity that allows the prediction of the stereochemistry of the hydride transfer for enoyl reductases of unknown specificity. Further considerations on the reaction mechanism indicated that crotonyl-CoA carboxylase/reductase may have evolved from enoyl-CoA reductases. This may be useful for protein engineering of enoyl reductases and their application in biocatalysis.

BIOCHEMISTRY

Communicated by Duilio Arigoni, Swiss Federal Institute of Technology, Zurich, Switzerland, April 12, 2009 (received for review December 9, 2008)

Fig. 1. Crotonyl-CoA carboxylase/reductase. Reactions catalyzed by Ccr. Properties of crotonyl-CoA carboxylase/reductase are shown in Table 1.

temperatures, depending on which species is used as the substrate (9, 10). In the case of Ccr, the oxidation of NADPH that corresponds to the formation of ethylmalonyl-CoA was faster when the reaction was started with dissolved CO2 compared with HCO⫺ 3, strongly indicating that CO2 is the carboxylating species in this reaction (Fig. 2). Addition of carbonic anhydrase to the reaction mixture led to identical reaction rates, independent of the CO2 species that was used in the reaction mixture. Because carbonic anhydrase catalyzes the reversible hydration of CO2 and therefore strongly increases the velocity of equilibration between dissolved CO2 and HCO⫺ 3 (11) that is limited under these conditions, these results support the contention that CO2 is the reactive species. Stereochemistry of the Carboxylation Products. The stereochemistry of the carboxyl group that is introduced at the C2 atom was elucidated by enzymatic analysis of methylmalonyl-CoA as the product of acryloyl-CoA carboxylation by Ccr. Incubation of Ccr with acryloyl-CoA, NADPH and 14C-labeled NaHCO3 yielded [3-carboxy-14C]-methylmalonyl-CoA (Fig. 3A) that was incubated subsequently with methylmalonyl-CoA epimerase (Epi) and/or (2R)-methylmalonyl-CoA mutase (Mcm). Methylmalonyl-CoA epimerase catalyzes the isomerization of (2S)-methylmalonyl-CoA and (2R)-methylmalonyl-CoA, whereas (2R)-methylmalonylCoA mutase catalyzes the coenzyme B12-dependent carbon skeleton rearrangement of (2R)-methylmalonyl-CoA to succinyl-CoA (Fig. S1) (12, 13). HPLC analysis showed succinyl-CoA formation only in the presence of both enzymes (Epi and Mcm), whereas incubation with either Epi or Mcm alone did not result in formation of succinyl-CoA (Fig. 3 B–D). We therefore conclude that carboxylation proceeds to the re face at the C2 atom (Fig. S2), resulting in products with (2S)-stereochemistry.

Fig. 2. Determination of the carboxylating species of the Ccr reaction. The oxidation of NADPH during the reductive carboxylation of crotonyl-CoA to ethylmalonyl-CoA was followed spectrophotometrically at 360 nm. To determine the carboxylating species, the reaction was started either with dissolved CO2 (black solid line), or HCO3⫺ (gray solid line) at 15 °C, a temperature, at which the hydration of CO2/dehydration of HCO3⫺ is slow. As control, the reaction was started also with dissolved CO2 (black dotted line) or HCO3⫺ (gray dotted line) in the presence of carbonic anhydrase (C.A.), an enzyme that catalyzes the reversible hydration of CO2.

this transfer, [2H]-(4R)- and [2H]-(4S)-NADPH were synthesized enzymatically and purified. The position of label was confirmed by NMR (see Fig. S3), and the labeled coenzymes were used in subsequent experiments. Incubation of crotonyl-CoA (m/z ⫽ 834) 2 in the presence of HCO⫺ 3 and NADPH or [ H]-(4S)-NADPH yielded ethylmalonyl-CoA with m/z ⫽ 880 as identified by HPLC-MS (Fig. 4 A and B). Conversely, incubation of crotonylCoA with [2H]-(4R)-NADPH resulted in ethylmalonyl-CoA of m/z ⫽ 881, indicating that a 2H-transfer had taken place (Fig. 4C). This demonstrated that the reductive carboxylation reaction of Ccr is pro-(4R) specific with respect to NADPH. Stereochemistry of the Hydride Transfer (Reductase Reaction). The

stereochemistry of the hydride transfer from NADPH was also investigated for the reduction reaction of Ccr that takes place in the absence of HCO⫺ 3 /CO2. Incubation of crotonyl-CoA with Ccr resulted under these conditions in butyryl-CoA of m/z ⫽ 836, when unlabeled NADPH (Fig. 5A) or [2H]-(4S)-NADPH was used, as shown by HPLC-MS. [2H]-(4R)-NADPH yielded butyryl-CoA with m/z ⫽ 837 (Fig. 5B), demonstrating that the reduction reaction of Ccr is pro-(4R) specific. The stereochemistry of the hydride transfer is thus conserved for both reactions catalyzed by Ccr.

Stereochemistry of the Hydride Transfer (Carboxylase Reaction). The

Cryptic Stereochemistry at the C3 Atom of the Product (Reductase Reaction). To investigate the stereochemistry of the hydride that is

hydride that is transferred from NADPH onto a substrate can be derived either from the pro-(4S) or pro-(4R) position at C4 of the nicotinamide ring (Fig. S2). To investigate the stereochemistry of

transferred to the C3 atom of the product, butyryl-CoA (labeled or unlabeled) was isolated from the reaction mixtures described above by preparative HPLC. These [2H]-C3-labeled butyryl-CoA and

Table 1. Properties of crotonyl-CoA carboxylase/reductase Parameter Reductive carboxylation Specific activity (Vmax) Substrates

Apparent Km values pH optimum Molecular composition Reduction Specific activity Apparent Km values §One

Recombinant crotonyl-CoA carboxylase/reductase 40 units§ mg⫺1 (this study, tagged enzyme 100 units mg⫺1 Crotonyl-CoA (100% relative specific activity),acryloyl-CoA (40%) Crotonyl-N-acetylcysteamine, methacryloyl-CoA, 6-hydroxycylohex-1-ene-1-carboxyl-CoA, cyclo-hexa-1,5-diene-1-carboxyl-CoA, acetoacetyl-CoA, propionyl-CoA, (R)-3-hydroxybutyryl-CoA, (S)-3-hydroxybutyryl-CoA (all ⬍1%) 0.4 mM crotonyl-CoA (0.5 mM acryloyl-CoA); 0.7 mM NADPH; 14 mM HCO3⫺ (equivalent to 0.2 mM ⬙dissolved CO2⬙ at pH 7.8) 7.5–8.0 Native molecular mass: 105 ⫹ 11 kDa, subunit molecular mass: 47 kDa 3 units mg⫺1 (this study, tagged enzyme) 10 units mg⫺1 (according to ref. 8, nontagged enzyme) 0.2 mM crotonyl-CoA; NADPH not determined

unit corresponds to 1 mmol of product formed per min.

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Erb et al.

Determination of the Stereospecificity of the Solvent Proton Addition (Reductase Reaction). To determine the stereochemistry of the

solvent proton that is added at the C2 position of crotonyl-CoA, the reduction of crotonyl-CoA to butyryl-CoA was performed in [2H]2O in the absence of HCO⫺ 3 /CO2. Incubation of crotonyl-CoA

Fig. 4. Determination of the stereochemistry of the hydride transfer (carboxylase reaction). The stereospecificity of the hydride transfer from the prochiral C4 position of the nicotinamide was determined by using stereospecifically labeled NADPH. Crotonyl-CoA was incubated in the presence of HCO3⫺/CO2 with unlabeled NADPH (A), [2H]-(4S)-NADPH (B), or [2H]-(4R)NADPH (C). The products were analyzed by HPLC-MS. The corresponding mass spectra of the ethylmalonyl-CoA species formed are shown in detail.

Erb et al.

with NADPH in [2H]2O yielded butyryl-CoA at a predominant m/z of 837, indicating that a 2H of the solvent had been incorporated (Fig. 5C). HPLC-MS also showed that ⬇10% of butyryl-CoA with a m/z of 836 were present. This corresponds exactly to the amount of unlabeled H2O (10% vol/vol) that is brought into the reaction mixture by the aqueous Ccr solution. Consequently, incubation of crotonyl-CoA with [2H]-(4R)-NADPH in [2H]2O resulted in butyryl-CoA with m/z ⫽ 838 and ⬇10% of m/z ⫽ 837 (Fig. 5D). [2H]-C2- and [2H2]-C2,C3-double-labeled butyryl-CoA that had been isolated by preparative HPLC were both transformed by butyryl-CoA dehydrogenase to crotonyl-CoA with a predominant m/z ⫽ 834 (Fig. 5 C and D), which means that in both cases, the deuterium atoms had been lost. Because butyryl-CoA dehydrogenase specifically removes the pro-(3R)- and pro-(2R)-hydrogen atoms of butyryl-CoA, these results clearly show that incorporation of the solvent hydrogen occurs predominantly at C2 to the re face (Fig. S2) and therefore anti to the hydride from NADPH that is transferred onto C3. Interestingly, when butyryl-CoA was labeled at the C2 atom, part of the label (⬇25%) was not removed by butyryl-CoA dehydrogenase as expected for an enzymatic reaction. This indicates that the solvent proton is not incorporated 100% in the pro-(2R) position but that to a smaller extent, incorporation also occurs to the si face. Such a finding is not surprising considering the reductase reaction as a physiological nonrelevant ‘‘side reaction’’ of the enzyme when compared with the carboxylase reaction that is catalyzed 10 times faster by Ccr. The observed loss of stereocontrol in case of the reduction reaction may be due to the geometry of the active site that has evolved to direct the incorporation of a carboxyl PNAS 兩 June 2, 2009 兩 vol. 106 兩 no. 22 兩 8873

BIOCHEMISTRY

unlabeled butyryl-CoA species were incubated with butyryl-CoA dehydrogenase. This flavoenzyme stereospecifically removes the pro-(2R)- and pro-(3R)-hydrogen atoms of butyryl-CoA yielding crotonyl-CoA (14). In all cases, the products formed (crotonylCoA) had a m/z of 834, according to a loss of label during the oxidation of [2H]-C3 labeled butyryl-CoA, as determined by HPLC-MS (Fig. 5 A and B). This indicates that the hydride transferred from NADPH onto the C3 of butyryl-CoA occurs to the re face (Fig. S2) resulting in pro-(3R)-butyryl-CoA.

Fig. 5. Determination of the stereochemistry of the reductase reaction. (A and B) Stereochemistry at C3. Crotonyl-CoA was incubated in the absence of HCO3⫺]/ CO2 with Ccr and either NADPH or [2H]-(4R)-NADPH, and the butyryl-CoA species formed were analyzed by HPLC-MS. The corresponding HPLC chromatograms (A1, B1) are shown together with the detailed mass spectra of the respective butyryl-CoA peaks. The butyryl-CoA species were isolated by preparative HPLC from the reaction mixture and converted back to crotonyl-CoA by pro-(2R), pro-(3R)-specific butyryl-CoA dehydrogenase (BDH) from pig liver mitochondria, to determine the absolute stereochemistry of the label incorporated. The corresponding HPLC chromatograms (A2, B2) and the respective mass spectra of crotonyl-CoA species are shown. (C and D) Stereochemistry at C2. Crotonyl-CoA was incubated in the absence of HCO3⫺/CO2 in [2H]2O with Ccr and either unlabeled NADPH or [2H]-(4R)-NADPH and the butyryl-CoA species formed were analyzed by HPLC-MS. The corresponding HPLC chromatograms (C1, D1) and detailed mass spectra of the butyryl-CoA peaks are shown. After isolation of the butyryl-CoA species from the reaction mixtures by preparative HPLC, the CoAesters were converted back to crotonyl-CoA by butyryl-CoA dehydrogenase (BDH) to determine the absolute stereochemistry of the label incorporated. The HPLC chromatograms (C2, D2) and mass spectra of crotonyl-CoA species are shown in detail.

CHEMISTRY

Fig. 3. Determination of the stereochemistry of the carboxylation product. (A) Acryloyl-CoA was incubated with NADPH, Ccr, and 14C-labeled NaHCO3, resulting in radioactive-labeled methylmalonyl-CoA as shown by HPLC and radioactive monitoring. (B–D) To determine the stereochemistry of the product, this methylmalonyl-CoA was subsequently incubated for 1 min with methylmalonyl-CoA epimerase (Epi) (B), (2R)-methylmalonyl-CoA mutase (Mcm) (C), or a combination of both enzymes (D). The formation of radioactive labeled products was followed by HPLC.

group that is electronically and sterically different from a (solvent) proton. Discussion In this study, the reactions catalyzed by crotonyl-CoA carboxylase/ reductase have been investigated in detail, and the complete stereochemical course of both reactions has been elucidated. The reduction of crotonyl-CoA to butyryl-CoA occurs from pro-(4R)NADPH onto the re face of the C3 atom of crotonyl-CoA, and the solvent proton is added mainly in anti fashion to the re face at C2 (Scheme 1). Correspondingly, in case of the reductive carboxylation of crotonyl-CoA to ethylmalonyl-CoA, the carboxyl group is also incorporated to the re face, resulting in products with (2S)stereochemistry (Fig. S2). Functional and Historical Models of Stereospecificity in Reduction Reactions. The stereochemistry of NAD(P)H dependent reductases/

dehydrogenases has been investigated in detail for ⬎50 years (15), and it has been shown in very early studies on ⬎100 dehydrogenases that approximately half of the enzymes catalyze a transfer of the pro-(4R) hydrogen and the other half transfer the pro-(4S)hydrogen (16). Moreover, crystal structures have shown that pro(4R)-specific dehydrogenases bind the NAD(P)H cofactor in anti conformation, whereas binding of the cofactor in pro-(4S)-specific dehydrogenases occurs in syn conformation (see Fig. S2) (17, 18). Ab initio molecular orbital calculations agree that for the anti conformation of NAD(P)H, the transfer of the pro-(4R) hydrogen is preferred, whereas the pro-(4S) hydrogen is transferred preferentially if the NAD(P)H is bound in syn conformation (18), which in each case corresponds to a transfer of the hydride ion from the pseudoaxial position. It has been argued further, that NAD(P)H bound in anti conformation is a weaker reducing agent than NADPH bound in syn and that optimal enzymes bind substrates so as to match the free energies of the bound intermediates (16). Because of striking correlations of the stereospecificity of reductases/dehydrogenases and the change in free energy between their corresponding substrates and products, a functional model has been proposed in which the nature of the stereospecificity [pro-(4R) or pro-(4S)] is dictated by a mechanistic imperative (16). However, some exceptions have been reported in which enzymes that accept the same substrates show different stereospecificities (19), and these findings are difficult to explain by a functional model. Thus, recent analyses that take amino acid sequences into account favor a historical model, in which stereospecificity is a nonselected trait that is conserved during divergent evolution (19–21). According to this model, enzymes within a specific class that are related on the amino acid sequence level all catalyze their respective reactions with the same stereospecificity, whereas enzymes that are nonhomologous, and thus differ in their amino acid sequence, may catalyze reactions with opposite stereochemistry (20). Enoyl (-thioester) reductases display an interesting case in which both a functional and a historical model are considered to explain the stereochemistry observed. In all cases examined, the addition of the solvent proton occurs at C2. This regioselectivity is consistent with a functional model due to the strong polarization of the ␣–␤ unsaturated double bond (20). In contrast, the observed diversity in stereoselectivity of the addition at C2 and C3 has been correlated to distinct amino acid sequences that reflect different evolutionary origins of the respective enzymes (20). Stereospecificity of Ccr and Related Reductases/Dehydrogenases. Ccr is distantly related to proteins of the medium-chain dehydrogenase/ reductase superfamily, among which are alcohol dehydrogenase from horse liver (amino acid sequence identity/similarity: 22/36%) (22), ␨-crystallin from human lens (23/39%) (23), and quinone oxidoreductase from Escherichia coli (24/40%) (24), as well as Thermus thermophilus HB8 (24/39%) (25). The crystal structures of all these proteins have shown that binding of the NAD(P)H 8874 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0903939106

cofactor occurs in anti conformation with several interactions of the pro-(4S) face of NAD(P)H to amino acids that are responsible for the binding of the nicotinamide, but not involved in catalysis. These steric interactions are in line with a transfer of the pro-(4R) hydride from NAD(P)H onto the respective substrate. In the case of horse liver alcohol dehydrogenase the pro-(4R) specificity of the hydride transfer has been experimentally verified (26). Altogether, these findings are well in line with the historical model, in which enzymes of a common origin and therefore of sequence similarity are supposed to catalyze reactions with the same stereospecificity, even though their substrates or the reduction reactions catalyzed (CAO reduction, CAC reduction, or reductive carboxylation) may differ quite considerably. A crotonyl-CoA reductase that is highly similar to Ccr from R. sphaeroides has been isolated from Streptomyces collinus (amino acid identity/similarity 41/56%), and its stereochemistry was studied in detail (27). The stereochemical course of hydride transfer and proton addition at crotonyl-CoA of the S. collinus enzyme corresponds to that defined for Ccr. By contrast, the hydride transfer that is pro-(4R) specific in case of Ccr was reported to be pro-(4S) specific for the enzyme from S. collinus. This is especially notable because of the high sequence similarity of the S. collinus enzyme and Ccr, which suggests that the S. collinus enzyme may represent a bona fide crotonyl-CoA carboxylase/reductase. This is further substantiated by the fact that reductive crotonyl-CoA carboxylation occurs in Streptomyces coelicolor A3 (2), a close relative of S. collinus (8), and that this activity is catalyzed by an enzyme of 93% amino acid sequence identity to the S. collinus enzyme (55). An explanation may be found in the original authors’ use of glucose dehydrogenase from Thermoplasma acidophilum to synthesize radioactive labeled (4S)-NADPH for the investigation of hydrogen transfer stereospecificity (27). Glucose dehydrogenase from T. acidophilum has since been demonstrated to be pro-(4R) specific, rather than pro-(4S) specific as originally assumed (28). Reevaluation of the original data therefore clearly shows that the S. collinus enzyme is actually pro-(4R) specific. This result is in good agreement with the conserved stereochemistry of the hydride transfer from the pro(4R) position of NADPH that is observed within the superfamily of medium-chain dehydrogenases/reductases that includes Ccr and the S. collinus enzyme, a fact that had been overlooked for the latter one thus far (20, 27). Interestingly, the enoyl thioester reductase domains of rat liver and chicken fatty acid synthases that have been reported to be pro-(4R) specific are also related to the same medium-chain dehydrogenase/reductase family (29, 30). The S. collinus enzyme and Ccr, which share the same nucleotide cofactor stereospecificity, may therefore also be related on evolutionary grounds to those enoyl reductase domains and may not have a different evolutionary origin as initially proposed (20, 27). General Pattern of Stereospecificity for Enoyl-(Thioester) Reductases (ER). Analysis of ERs, whose stereochemistry has been studied and

the amino acid sequence is available, shows a clear correlation of cofactor stereospecificity and domain architecture (Table S2, Fig. S4). Those ERs belonging to the family of medium-chain dehydrogenases/reductases (31) contain domains of the ADH㛭zinc㛭N superfamily (pfam00107) and are all pro-(4R) specific. Examples are ER domains of type I fatty acid synthase from rat liver and chicken (26), the type II fatty acid synthase ER Etr1p from the yeast Candida tropicalis (32), or alkenal/one oxidoreductase from rat (33). By contrast, the pro-(4S)-specific enzymes either contain domains of the AdoHcyase superfamily (cl09931) like FabI and InhA (both type II fatty acid synthase) from E. coli (26) and Mycobacterium tuberculosis (34), or of the TIM㛭phosphate㛭binding superfamily (cl09108) like the ER domains of type I fatty acid synthase from baker’s yeast (26) or 2,4-dienoyl-CoA reductase from E. coli (35). These correlations can be extended to the alcohol dehydrogenases of the medium-chain dehydrogenase/reductase family (horse liver, yeast and Pseudomonas fluorescens) that are all Erb et al.

Possible Reaction Mechanism for Ccr. Investigation of the carboxy-

lation reaction of Ccr showed that CO2 and not HCO⫺ 3 is the carboxylating species. In principle, CO2 can serve as electrophile in COC forming reactions on negatively polarized (or inverted) carbon atoms (5), whereas HCO⫺ 3 is a nucleophile that has to be transformed into the carboxylating species by formation of a reactive carboxyphosphate upon ATP/phosphoester hydrolysis or as carboxyl-biotin to serve in enzymatic carboxylation reactions (44, 45). Because the carboxylation reaction of Ccr is neither ATP- nor biotin-dependent (8), this further supports the role of CO2 as a carboxylating species. Dissection of the reductive carboxylation into 2 separate ‘‘half reactions’’ shows that reduction of crotonyl-CoA to butyryl-CoA is an exergonic process, and that the free-energy change associated with this reduction should be able to drive the endergonic carboxylation reaction (46). Considering the experimental results, we therefore propose the following reaction mechanism for crotonyl-CoA carboxylase/reductase.‡ First, a hydride ion is transferred from NADPH onto C3 of crotonyl-CoA to give a thioester enol(ate), followed by an electrophilic attack of the CO2 at C2. These chemical steps may occur either simultaneously, as in a concerted mechanism, or successively, as in a stepwise mechanism. This cannot be differentiated by the current data. A small isotopic effect of ⬇1.7 on the kcat of the carboxylation reaction was †‘‘Trans-acting’’

ERs are accessory enzymes that interact with large multidomain enzymes (e.g. type I PKS) to complement the activity of a missing (or inactive) ER domain.

‡This

model is based on a proposal from W. Buckel at the combined seminar of the Graduiertenkolleg ‘‘Protein function on atomic level’’ (Marburg, Germany) and ‘‘Biochemistry of the enzymes’’ (Freiburg, Germany) 2007 in Hirschegg, Austria.

Erb et al.

observed when [2H]-(4R)-NADPH was used instead of unlabeled NADPH. This suggests that the hydride transfer onto the substrate is partially involved in the rate-limiting step of the reaction (47). A stepwise mechanism has been demonstrated for 2,4-dienoylCoA reductase from rat liver mitochondria that catalyzes the NADPH-dependent reduction of 2,4-dienoyl-CoA thioesters to the resulting trans-3-enoyl-CoA products. The formation of a dienolate anion intermediate during the course of reaction has been shown by kinetic and spectrophotometric methods (48). Similarly, incubation of Ccr with crotonyl-CoA in the absence of CO2 may also result in the formation of such an enolate anion, because the true electrophile CO2 is missing, and the rate-limiting step is shifted to the addition of a solvent proton replacing that CO2 molecule. This speculation may be supported by the observation that, in contrast to the carboxylation reaction, no isotopic effect on the kcat of the reduction reaction was observed when [2H]-(4R)-NADPH was used instead of unlabeled NADPH. mechanistic model of the reactions catalyzed by Ccr with implications on the evolution of this enzyme class. Comparison of amino acid sequences of Ccr and well-studied members of the mediumchain dehydrogenase/reductase superfamily indicate that all enzymes share the same origin (49). One might therefore speculate that crotonyl-CoA carboxylase/reductase has emerged from reductases and that it has evolved an active center that clearly prefers the carboxylation of crotonyl-CoA over the reduction reaction. From this point of view, the reduction reaction of Ccr may be taken as an evolutionary relict, resulting in a much lower catalytic efficiency as well as less conserved stereochemistry at C2 compared with the carboxylation. In summary, the evolution of a primordial enoylCoA reductase toward a true carboxylase may provide the basis for further protein engineering of enoate reductases. Identification of those residues and/or structural properties that direct the carboxylation reaction and are involved in the preferential binding of a CO2 molecule at the active site may in turn allow a rational design of enoate carboxylases (or enzymes that incorporate other electrophiles) on the scaffold of already known enoate reductases and would extend the synthetic toolbox of organic chemists. Materials and Methods Preparation of Enzymes and Substrates. Recombinant Ccr was produced in 200-L scale and purified from cell extracts (15 mL, 1.6 g of protein) as described (8). Epi and Mcm were prepared as described previously (50). A histidine-tagged version of Ccr(Ccrhis) was produced as described in detail in SI Methods. Butyryl-CoA dehydrogenase was prepared from pig liver as described (51, 52) with minor modifications (see SI Methods). Crotonyl-CoA and acryloyl-CoA were synthesized as reported before (50). [2H]-(4R)- and [2H]-(4S)-NADPH were synthesized and purified according to Pollock and Barber (53) with some modifications (see SI Methods for details). The purified products were characterized by NMR (Fig. S3). Determination of the Carboxylating Species. The active species of CO2 was determined spectrophotometrically after a modified method (9) in a cuvette (d ⫽ 0.1 cm) at 15 °C, following the rate of NADPH oxidation at 360 nm (␧NADPH ⫽ 3.4 mM⫺1 cm⫺1). For experimental details, see SI Methods. Stereochemistry of the Carboxylation Product. Radioactive-labeled methylmalonyl-CoA was synthesized from acryloyl-CoA and H14CO3 by Ccr and subsequently used as substrate for different combinations of Epi and/or Mcm as described recently (50). Samples were withdrawn from the reaction mixtures and analyzed subsequently by HPLC and radioactive monitoring (see below). Further details are described in SI Methods. Stereospecificity of the Hydrogen Transfer from NADPH (Carboxylase Reaction). Ethylmalonyl-CoA species were synthesized from crotonyl-CoA and NaHCO3 by Ccr as described before (8) by using [2H]-(4R)-NADPH, [2H]-(4S)-NADPH or unlabeled NADPH, respectively. The ethylmalonyl-CoA formed was analyzed by HPLC-MS (see below, SI Methods). PNAS 兩 June 2, 2009 兩 vol. 106 兩 no. 22 兩 8875

BIOCHEMISTRY

Concluding Remarks. The results of this study suggest a detailed

CHEMISTRY

pro-(4R) specific and harbor an ADH㛭zinc㛭N superfamily domain. Similarly, Drosophila (short chain) alcohol dehydrogenase and the keto-acyl carrier protein reductase Sco1815 (type II polyketide synthase, PKS) involved in R1128 biosynthesis from S. coelicolor (36) are both pro-(4S) specific and possess a domain of the AdoHcyase superfamily. For alcohol (polyol) dehydrogenases, such correlations have actually been anticipated (37), and our results on ER therefore strongly support the assumption that the cofactor stereospecificity is preserved within structural defined enzymatic domains. When ERs of unknown stereoselectivities are analyzed, their stereochemistry can be assigned according to Table S2 (see also Fig. S4), if they contain 1 of the 3 domains described above. We therefore propose that ER domains of the type I PKS are pro-(4R) specific, as deduced for the respective domains of the Nystatin and Epothilone biosynthesis modules. In keeping with this, the ER LovC that acts in trans† on the (iterative) type I lovastatin PKS (38) also displays pro-(4R) stereospecificity, whereas Rv2953 that similarly operates in trans during the biosynthesis of (phenol)glycolipids in M. tuberculosis (39) cannot be assigned on the basis of its domain structure. However, further sequence–distance relationship analysis indicate that Rv2953 may be pro-(4S) specific (Fig. S4). ERs like FabK (40), FabL (41), or FabV (42) that replace the widely distributed FabI component of fatty acid synthases (type II) in a number of bacteria, most likely are pro-(4S) specific according to Table S2. It is noteworthy to mention that, to date, any experimental evidence for the stereospecificity of PKS ER (domains) is missing (43). This is also the case for ERs that substitute FabI in bacterial fatty acid synthesis. Clearly, further experimentation is required to prove the suggested correlation of cofactor stereospecificity and domain architecture of ERs. However, a similar correlation between substrate stereospecificity and amino acid sequence cannot be drawn easily, because the substrate that is bound to the active site as well as the reactions that are catalyzed differ quite remarkably. Therefore, the prediction of the complete stereochemistry of an enzymatic reaction by analysis of the amino acid sequence or the domain structure remains challenging.

Stereospecificity of the Hydrogen Transfer from NADPH (Reductase Reaction). Butyryl-CoA species were synthesized from crotonyl-CoA by Ccr as described above, by using [2H]-(4R)-NADPH, [2H]-(4S)-NADPH, or unlabeled NADPH and omitting NaHCO3 from the reaction mixture. The butyryl-CoA formed was analyzed by HPLC-MS (see below, SI Methods). Cryptic Stereochemistry at C3 (Reductase Reaction). [2H]-C3-butyryl-CoA (synthesized from [2H]-(4R)-NADPH) and butyryl-CoA (synthesized from NADPH) were purified by preparative HPLC and incubated with butyryl-CoA dehydrogenase preparations in a modified assay (54) by using ferricenium hexafluorophosphate as electron acceptor. The crotonyl-CoA formed was analyzed by HPLC-MS (see below, SI Methods).

solvent. [2H]-C2-butyryl-CoA (synthesized from NADPH in [2H]2O) and [2H2]C2,C3-butyryl-CoA (synthesized from [2H]-(4R)-NADPH in [2H]2O) were isolated by preparative HPLC and subjected to butyryl-CoA dehydrogenase as described above. Miscellaneous. All CoA esters were analyzed by reversed-phase HPLC(-MS) by using UV detection and radioactive monitoring as described recently (8, 50). For details, see SI Methods. Accession numbers and amino acid sequences of the proteins analyzed in this study are in Tables S3 and S4, respectively.

Stereoselectivity of the Solvent Hydrogen Addition (Reductase Reaction). Reduction of crotonyl-CoA by Ccr was performed as described by using [2H]2O as

ACKNOWLEDGMENTS. We thank D.A. for excellent comments on the manuscript, Frederik Golitsch for technical assistance, and Ivan Berg and Toma´sˇ and Hana Sˇmejkal for critical reading. This work was supported by Deutsche Forschungsgemeinschaft Grant AL677/1-1, Evonik Industries AG, and Fonds der Chemischen Industrie.

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28. Mostad SB, Helming HL, Groom C, Glasfeld A (1997) The stereospecificity of hydrogen transfer to NAD(P) catalyzed by lactol dehydrogenases. Biochem Biophys Res Comm 233:681– 686. 29. Leibundgut M, Maier T, Jenni S, Ban N (2008) The multienzyme architecture of eukaryotic fatty acid synthases. Curr Opin Struct Biol 18:714 –725. 30. Kwan DH, et al. (2008) Prediction and manipulation of the stereochemistry of enoylreduction in modular polyketide synthases. Chem Biol 15:1231–1240. 31. Persson B, Zigler JS, Jr, Jo¨rnvall H (1994) A superfamily of medium-chain dehydrogenases/reductases (MDR)—Sub-lines including ␨-crystallin, alcohol and polyol dehydrogenases, quinone oxidoreductases, enoyl reductases, VAT-1 and other proteins. Eur J Biochem 226:15–22. 32. Airenne TT, et al. (2003) Structure–function analysis of enoyl thioester reductase involved in mitochondrial maintenance. J Mol Biol 327:47–59. 33. Dick RA, Kensler TW (2004) The catalytic and kinetic mechanisms of NADPH-dependent alkenal/one oxidoreductase. J Biol Chem 279:17269 –17277. 34. Bell AF, et al. (2007) Evidence from Raman spectroscopy that InhA, the mycobacterial enoyl reductase, modulates the conformation of the NADH cofactor to promote catalysis. J Am Chem Soc 129:6425– 6431. 35. Fillgrove KL, Anderson VE (2000) Orientation of coenzyme A substrates, nicotinamide and active site functional groups in (di)enoyl-coenzyme A reductases. Biochemistry 39:7001–7011. 36. Tang YY, et al. (2006) Structural and functional studies on SCO1815: A beta-ketoacylacyl carrier protein reductase from Streptomyces coelicolor A3(2). Biochemistry 45:14085–14093. 37. Schneider-Bernlo¨hr H, Adolph HW, Zeppezauer M (1986) Coenzyme stereospecificity of alcohol/polyol dehydrogenases: Conservation of protein types vs. functional constraints. J Am Chem Soc 108:5573–5576. 38. Kennedy J, et al. (1999) Modulation of polyketide synthase activity by accessory proteins during lovastatin biosynthesis. Science 284:1368 –1372. 39. Simeóne R, Constant P, Guilhot C, Daffe´ M, Chalut C (2007) Identification of the missing trans-acting enoyl reductase required for phthiocerol dimycocerosate and phenolglycolipid biosynthesis in Mycobacterium tuberculosis. J Bacteriol 189:4597– 4602. 40. Heath RJ, Rock CO (2000) A triclosan-resistant bacterial enzyme. Nature 406:145–146. 41. Heath RJ, Su N, Murphy CK, Rock CO (2000) The enoyl-[acyl-carrier-protein] reductases FabI and FabL from Bacillus subtilis. J Biol Chem 275:40128 – 40133. 42. Massengo-Tiasse´ RP, Cronan JE (2008) Vibrio cholerae FabV defines a new class of enoyl-acyl carrier protein reductase. J Biol Chem 283:1308 –1316. 43. Smith S, Tsai S-C (2007) The type I fatty acid and polyketide synthases: A tale of two megasynthases. Nat Prod Rep 24:1041–1072. 44. Lynen F (1967) The role of biotin-dependent carboxylations in biosynthetic reactions. Biochem J 102:381– 400. 45. Knowles JR (1989) The mechanism of biotin dependent enzymes. Annu Rev Biochem 58:195–221. 46. Li F, et al. (2008) Coupled ferredoxin and crotonyl coenzyme A (CoA) reduction with NADH catalyzed by the butyryl-CoA dehydrogenase/Etf complex from Clostridium kluyveri. J Bacteriol 190:843– 850. 47. Walsh, C (1979) in Enzymatic Reaction Mechanism (Freeman, San Francisco), pp 109 –123. 48. Fillgrove KL, Anderson VE (2001) The mechanism of dienoyl-CoA reduction by 2,4dienoyl-CoA reductase is stepwise: Observation of a dienolate intermediate. Biochemistry 40:12412–12421. 49. Nordling E, Jo¨rnvall H, Persson B (2002) Medium-chain dehydrogenases/reductases (MDR). Family characterizations including genome comparisons and active site modeling. Eur J Biochem 269:4267– 4276. 50. Erb TJ, Re´tey J, Fuchs G, Alber BE (2008) Ethylmalonyl-CoA mutase from Rhodobacter sphaeroides defines a new subclade of coenzyme B12-dependent acyl-CoA mutases. J Biol Chem 283:32283–32293. 51. Shaw LL, Engel PC (1984) The purification and properties of ox liver short-chain acyl-CoA dehydrogenase. Biochem J 218:511–520. 52. Lundberg NN, Thorpe C (1993) Inactivation of short-chain acyl-coenzyme-A dehydrogenase from pig liver by 2-pentenoyl-coenzyme-A. Arch Biochem Biophys 305:454 – 459. 53. Pollock VV, Barber MJ (2001) Kinetic and mechanistic properties of biotin sulfoxide reductase. Biochemistry 40:1430 –1440. 54. Lehman TC, Thorpe C (1990) Alternate electron acceptors for medium-chain acyl-CoA dehydrogenase: Use of ferricenium salts. Biochemistry 29:10594 –10602. 55. Erb T (2009) The ethylmalonyl-CoA pathway: A novel acetyl-CoA assimulation strategy. Ph.D. thesis (Univ of Freiburg, Germany).

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Supporting Information Erb et al. 10.1073/pnas.0903939106 SI Methods Materials. Chemicals were obtained from Sigma–Aldrich, Merck, Applichem, Roth, or Gerbu. Materials for cloning and expression were purchased from MBI Fermentas, Novagen, Genaxxon Bioscience, biomers.net, or Qiagen. Materials and equipment for protein purification were obtained from GE Healthcare or Millipore. NaH14CO3 was from Hartmann Analytic. 2-Propanol-d8 (99.5 atom%), D-glucose-1-d (97 atom%), [2H]2O (99.98 atom%), glucose-6-phosphate dehydrogenase from baker’s yeast (type VII), alcohol dehydrogenase from Thermoanaerobium brockii, and carbonic anhydrase from bovine erythrocytes were purchased from Sigma-Aldrich. Preparation of Enzymes. Recombinant crotonyl-CoA carboxylase/ reductase (Ccr) was produced in 200-L scale and purified from cell extracts (15 mL, 1.6 g of protein) as described (16). Methylmalonyl-CoA epimerase (Epi) and (2R)-methylmalonyl-CoA mutase (Mcm) were prepared as recombinant histidinetagged (his-tagged) promiscuous ethylmalonyl-CoA/methylmalonyl-CoA epimerase and his-tagged methylmalonyl-CoA mutase as described previously (20). A his-tagged version of Ccr(Ccrhis) was produced by amplifying the gene encoding Ccr by PCR from R. sphaeroides chromosomal DNA using the forward primer (5⬘-GGA GGC AAC CAT GGC CCT CGA CGT GCA GAG-3⬘) introducing a NcoI site (italicized) at the initiation codon and reverse primer (5⬘-GAG ACT TGC GGA TCC CTC CGA TCA GGC CTT GC-3⬘) introducing a BamHI site (italicized) after the stop codon. The PCR product was isolated and cloned into the expression vector pRSET-B (Invitrogen), generating pTE42. Recombinant Ccrhis was produced in E. coli BL21(DE3) or Rosetta 2 (DE3) that had been transformed with pTE42. The cells were grown at 37 °C in LB medium with 100 ␮g mL⫺1 ampicillin. Expression was induced at an OD578 ⫽ 0.6–0.9 with 0.5 mM isopropyl-thiogalacto-pyranoside, the temperature was lowered to 30 °C, and the cells were harvested after additional growth for 3 h. Cells (1 g) were suspended in 2 mL of 20 mM Tris(hydroxymethyl)aminomethane Tris䡠HCl (pH 7.8) containing 0.1 mg ␮L⫺1 of DNase I, the suspension was passed twice through a chilled French pressure cell at 137 Mpa, and the cell lysate was centrifuged (100,000 g) at 4 °C for 1 h. An aliquot of the supernatant (2 mL, ⬇60 mg of protein) was applied at a flow rate of 1 mL min⫺1 onto a 1-mL Ni-Sepharose Fast Flow Column (HisTrap FF; Amersham) that had been equilibrated with 10 volumes of buffer A containing 20 mM Tris䡠HCl (pH 7.8) and 200 mM KCl. The column was washed with buffer A and buffer A containing 75 mM imidazole. Ccrhis was eluted with buffer A containing 500 mM imidazole. The enzyme was desalted and concentrated by ultrafiltration (Amicon YM 10 membrane; Millipore). The protein (3 mg) was stored at ⫺20 °C in 10 mM Tris䡠HCl (pH 7.8) with 50% glycerol. Butyryl-CoA dehydrogenase was prepared from pig liver as described (21, 22) yielding 30.5 g of mitochondria (wet weight) from 500 g of liver. Isolated mitochondria (6.5 g) were sonicated, and the crude extract was fractionated with (NH4)2SO4. Protein precipitated between 40% and 57% (NH4)2SO4 saturation was resuspended in 0.5 mL of 20 mM potassium phosphate buffer (pH 6.5) and dialyzed 2 times for 1.5 h against 2 L of the same buffer. One third of the nondiffusible material was applied onto a Sephadex A-50 column (16 mL) that had been equilibrated by the passage of 10 volumes of buffer B containing 25 mM potassium phosphate (pH 7.6) at a flow rate of 1 mL min⫺1. The Erb et al. www.pnas.org/cgi/content/short/0903939106

column was washed with 30 mL of buffer B, followed by 20 mL of buffer B with 50 mM KCl, a linear gradient from 50 mM to 250 mM KCl in buffer B over 40 mL, and a linear gradient from 250 to 500 mM KCl in buffer B over 10 mL. Fractions were collected and tested for the oxidation of butyryl-CoA (4). Active fractions that eluted at 200–400 mM KCl were pooled, desalted, and concentrated to a final volume of 1 mL by ultrafiltration (YM 10 membrane; Amicon). The protein (2 mg) was stored at ⫺20 °C in 10 mM Tris䡠HCl (pH 7.8) containing 50% glycerol. Syntheses. Crotonyl-CoA was synthesized from its anhydride (23)

and acryloyl-CoA was synthesized from the free acid by the method of Stadtman (24). Both CoA-esters were quantified by absorption at 260 nm (␧ ⫽ 22,000 M⫺1 cm⫺1) (25), and the purity was analyzed by a previously described HPLC method (16). [2H]-(4R)- and [2H]-(4S)-NADPH were synthesized according to Pollock and Barber (26) with some modifications. [2H](4R) NADPH was synthesized from 18 mg NADP⫹, 0.6 mL of 2-propanol-d8, and 38 units of alcohol dehydrogenase (Thermoanaerobium brockii), dissolved in 7.5 mL 25 mM Tris䡠HCl buffer (pH 9) at 42 °C. [2H]-(4S)-NADPH was synthesized from 18 mg of NADP⫹, 10 mg of D-glucose-1-d, 0.85 mL of dimethyl sulfoxide and 50 units of glucose-6-phosphate dehydrogenase (Saccharomyces cerevisiae), dissolved in 83 mM potassium phosphate buffer (pH 8) at 30 °C. Both reactions were followed spectrophotometrically at 340 nm until no further increase in absorbance was observed. The respective [2H]-NADPH stereoisomers were purified individually. To precipitate the reduced nucleotide, 12 volumes of ice-cold ethanol were added, and the solution was incubated for 20 min at ⫺20 °C. After centrifugation for 30 min at 27,000 ⫻ g, the supernatant was discarded, and the yellowish pellet was dissolved in 2 mL of H2O. The [2H]NADPH solution was applied onto a Whatman DE23 column (5 mL) that had been equilibrated with 10 volumes of H2O at a flow rate of 2 mL min⫺1. The column was washed with 40 mL of H2O, followed by a linear gradient from 0 to 0.5 M NH4HCO3 over 120 mL, and the reduced nucleotide eluted at 0.16 – 0.33 M NH4HCO3. The eluate was evaporated to remove NH4HCO3, the aqueous solution was lyophilized, and the product was stored at ⫺20 °C. To follow the synthesis and purification, each step was controlled by recording a spectrum of the respective solution and determining the absorbance ratio of A260/A340. The purified products were also characterized by NMR (Fig. S3). Determination of the Carboxylating Species. The active species of

CO2 was determined spectrophotometrically after a modified method (27) in a cuvette (d ⫽ 0.1 cm) at 15 °C, following the rate of NADPH oxidation at 360 nm (␧NADPH ⫽ 3.4 mM⫺1 cm⫺1). All solutions were prepared freshly and stored on ice until they were used. The carboxylation reaction with ‘‘dissolved CO2’’ was measured in a reaction mixture (0.182 mL) containing 180 mM Tris䡠HCl (pH 7.9), 4.9 mM NADPH, 1.7 mM crotonyl-CoA, and 38 ␮g of Ccr. To start the carboxylation reaction, 20 ␮L of 50 mM KHCO3 were mixed with 6 ␮L of 1 M acetic acid to dissolve CO2 from bicarbonate by acidification, and this ‘‘CO2 solution’’ was immediately added to the reaction mixture in the cuvette. To follow the carboxylation reaction with ‘‘bicarbonate,’’ the reaction mixture in the cuvette contained 173 mM instead of 180 mM Tris䡠HCl (pH 7.9) and 33 mM acetic acid. The ‘‘bicarbonate’’ solution was prepared by mixing 20 ␮L of 50 mM KHCO3 with 6 ␮L of 200 mM Tris䡠HCl (pH 7.9) and was added immediately to the reaction mixture in the cuvette. As control, both reactions 1 of 13

were also performed in the presence of 0.3 mg of carbonic anhydrase in the reaction mixture. Determination of the Stereochemistry of the Carboxylation Product.

Radioactive-labeled methylmalonyl-CoA was synthesized from acryloyl-CoA and H14CO3 by Ccr and subsequently used as substrate for different combinations of Epi and/or Mcm (20). The substrate mixture (495 ␮L) contained 80 mM Tris䡠HCl (pH7.8), 3.7 mM NADPH, 1.9 mM acryloyl-CoA, 7.9 mM NaHCO3, 0.4 MBq mL⫺1 NaH14CO3, and 270 ␮g of Ccr. After incubation at 30 °C for 5 min, a sample of 100 ␮L was removed from the substrate mixture and added to 10 ␮L of 20% formic acid (t0 sample). Aliquots of 106 ␮L were added to ‘‘protein solutions’’ containing 0.10 ␮mol of Co2⫹, 0.07 ␮mol of coenzyme B12, and either 0.3 ␮g of Epi, 6.3 ␮g of Mcm, or a combination of both proteins. Samples were withdrawn after 1 min of incubation at 30 °C and added to 10 ␮L of 20% formic acid (t1 samples). All samples were centrifuged to remove denatured protein and analyzed subsequently by HPLC and radioactive monitoring (see below). Determination of the Stereospecificity of the Hydrogen Transfer from NADPH (Carboxylase Reaction). All reactions were performed in a

cuvette (d ⫽ 0.1 cm) at 30 °C and followed spectrophotometrically at 360 nm. Crotonyl-CoA (1.8 mM) was incubated for 5 min in 0.335 mL of 90 mM Tris䡠HCl buffer (pH 8) containing 30 mM NaHCO3 and 4.5 mM [2H]-(4R)-NADPH, [2H]-(4S)-NADPH, or unlabeled NADPH (16). A sample of 100 ␮L was withdrawn, and 400 ␮L of methanol were added (‘‘crotonyl-CoA-t0 sample’’). The carboxylation reaction was started by adding 6 ␮g of Ccr [in 5 ␮L of 10 mM Tris䡠HCl (pH 7.8) containing 5% glycerol] to the solution in the cuvette. A sample of 100 ␮L was withdrawn after 10 min, and 400 ␮L of methanol were added (‘‘ethylmalonyl-CoA-CCR sample’’). Methanol was evaporated in a Speedvac concentrator, and the samples were analyzed subsequently by HPLC-MS (see below). Determination of the Stereospecificity of the Hydrogen Transfer from NADPH (Reductase Reaction). All reactions were performed in a

cuvette (d ⫽ 0.1 cm) at 30 °C and monitored by a spectrophotometer at 360 nm. Crotonyl-CoA (2 mM) was incubated for 5 min in 0.3 mL of 40 mM Tris䡠HCl buffer (pH 7.6) containing 4 mM [2H]-(4R)-NADPH, [2H]-(4S)-NADPH, or unlabelled NADPH (16). A sample of 100 ␮L was withdrawn, and 10 ␮L of 20% HCOOH were added (‘‘crotonyl-CoA-t0 sample’’). The reduction reaction was started by adding 12 ␮g of Ccrhis [in 12 ␮L of 10 mM Tris䡠HCl (pH 7.8) containing 50% glycerol] to the solution in the cuvette. When no further decrease in the absorption was observed, 20 ␮L of 20% HCOOH were added to the reaction mixture (‘‘butyryl-CoA-CCR sample’’). All samples were centrifuged to remove denatured protein and analyzed subsequently by HPLC-MS (see below).

1. Tabita RF, et al. (2007) Function, structure, and evolution of the RubisCO-like proteins and their RubisCO homologs. Microbiol Mol Biol Rev 71:576 –599. 2. Chollet R, Vidal J, O’Leary MH (1996) Phosphoenolpyruvate carboxylase: A ubiquitous, highly regulated enzyme in plants. Annu Rev Mol Plant Biol 47:273–298. 3. Matte A, Tari LW, Goldie H, Delbaere LT (1997) Structure and mechanism of phosphoenolpyruvaste carboxykinase. J Biol Chem 272:8105– 8108. 4. Sluis MK, et al. (2002) Biochemical, molecular, and genetic analyses of the acetone carboxylases from Xanthobacter autotrophicus strain Py2 and Rhodobacter capsulatus strain B10. J Bacteriol 184:2969 –2977. 5. Jobst B (2005) Biochemie der Acetophenon Carboxylase, eines Schlu¨sselenzyms des anaeroben Ethylbenzol-Stoffwechsels. PhD thesis (University of Freiburg, Freiburg, Germany). 6. Jitrapakdee S, et al. (2008) Structure, mechanism and regulation of pyruvate carboxylase. Biochem J 413:369 –387.

Erb et al. www.pnas.org/cgi/content/short/0903939106

Determination of the Cryptic Stereochemistry at C3 (Reductase Reaction). [2H]-C3-butyryl-CoA (synthesized from [2H]-(4R)-

NADPH) and butyryl-CoA (synthesized from NADPH) were purified by preparative HPLC from 100 ␮L of a ‘‘butyryl-CoA sample’’ (see above), followed by lyophilization. The CoA esters isolated were each dissolved in 194 ␮L of 60 mM Tris䡠HCl (pH 7.8) containing 2.7 mM ferrocenium hexafluorophosphate (30). The solutions were transferred into a cuvette (d ⫽ 0.1 cm), and all reactions were followed spectrophotometrically at 300 nm and 30 °C. The dehydrogenase reaction was started by adding 20–40 ␮g of protein of the butyryl-CoA dehydrogenase preparation (see above) to the cuvette and 20 ␮L of 20% HCOOH were added after 15–20 min (‘‘crotonyl-CoA-BDH sample’’) to stop the reaction. The samples were centrifuged to remove denatured protein and subsequently analyzed by HPLC-MS (see below). Determination of the Stereoselectivity of the Solvent Hydrogen Addition (Reductase Reaction). Reduction of crotonyl-CoA by Ccr

was performed as described using [2H]2O instead of water. [2H]-C2-butyryl-CoA (synthesized from NADPH in [2H]2O) and [2H2]-C2,C3-butyryl-CoA (synthesized from [2H]-(4R)-NADPH in [2H]2O) were isolated and subjected to butyryl-CoA dehydrogenase as described above. Analysis of CoA Esters by HPLC and HPLC-MS. All CoA esters were separated by reversed-phase HPLC on a C18 column (LiChrospher 100, end-capped, 5 ␮m, 125 ⫻ 4 mm; Merck). Reaction products and standard compounds were detected by UV absorbance with a Waters 996 photodiode array detector. Radioactivity of eluting compounds was monitored by a Ramona 2000 radioactive monitor (Raytest) connected in series. For the separation of methylmalonyl-CoA and succinyl-CoA, the column was developed at a flow rate of 1 mL min⫺1 for 7 min under isocratic conditions with 100 mM NaH2PO4 (pH 4.0) in 7.5% methanol (vol/vol), followed by a linear 10-min gradient from 0 to 60% 100 mM sodium acetate (pH 4.2) in 90% methanol (vol/vol) (retention times: methylmalonyl-CoA, 11.7 min; succinyl-CoA, 12.3 min) (20). For the separation of crotonyl-CoA and ethylmalonyl-CoA, the column was developed at a flow rate of 1 mL min⫺1 by a linear gradient from 2% acetonitrile (CH3CN) in 50 mM ammonium acetate (pH 6.8) to 10% CH3CN within 30 min, followed by a linear gradient from 10% CH3CN to 45% CH3CN within 5 min (retention times: crotonyl-CoA, 10.1 min; ethylmalonyl-CoA, 22.0 min) (16). Crotonyl-CoA and butyrylCoA were separated at a flow rate of 1 mL min⫺1 for 2 min under isocratic conditions with 6% CH3CN in 50 mM ammonium acetate (pH 6.8), followed by a 17-min linear gradient from 6% CH3CN to 17% CH3CN and a 3-min linear gradient from 17% CH3CN to 45% (retention times: crotonyl-CoA, 10.7 min; butyryl-CoA, 13.6 min). HPLC-MS was performed on an Agilent 1100 system (Agilent Technologies) interfaced with an Applied Biosystems API 2000 triple-quadrupole spectrometer. The temperature of the Turbo-Ionspray auxiliary gas was 400 °C, and the ionization voltage was ⫺4,500 V. The samples were analyzed with a mass range of 100–1,600 Da. 7. Aoshima M, Igarashi Y (2006) A novel oxalosuccinate-forming enzyme involved in the reductive carboxylation of 2-oxoglutarate in Hydrogenobacter thermophilus TK-6. Mol Microbiol 62:748 –759. 8. Knowles JR (1989) The mechanism of biotin dependent enzymes. Annu Rev Biochem 58:195–221. 9. Aguilar JA, et al. (2008) Substrate specificity of the 3-methylcrotonyl coenzyme A (CoA) and geranyl-CoA carboxylases from Pseudomonas aeruginosa. J Bacteriol 190:4888 – 4893. 10. Rishavy MA, et al. (2004) A new model for vitamin K-dependent carboxylation: The catalytic base that deprotonates vitamin K hydroquinone is not Cys but an activated amine. Proc Natl Acad Sci USA 101:13732–13737. 11. Omura H, Wieser M, Nagasawa T (1998) Pyrrole-2-carboxylate decarboxylase from Bacillus megaterium PYR2910, an organic-acid-requiring enzyme. Eur J Biochem 253:480 – 484. 12. Thauer RK, Kaüfer B, Scherer P (1975) The active species of ‘‘CO2’’ utilized in ferredoxinlinked carboxylation reactions. Arch Microbiol 104:237–240.

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13. Schut GJ, Menon AL, Adams MW (2001) 2-keto acid oxidoreductases from Pyrococcus furiosus and Thermococcus litoralis. Methods Enzymol 331:144 –158. 14. Schu¨le K, Fuchs G (2004) Phenylphosphate synthase: A new C-C lyase involved in anaerobic phenol metabolism in Thauera aromatica. J Bacteriol 186:4556 – 4567. 15. Clark DD, Allen JR, Ensign SA (2000) Characterization of five catalytic activities associated with the NADPH:2-ketopropyl-coenzyme M oxidoreductase/carboxylase of the Xanthobacter strain Py2 epoxide carboxylase system. Biochemistry 39:1294 –1304. 16. Erb TJ, et al. (2007) Synthesis Of C5-dicarboxylic acids from C2-units involving crotonylCoA carboxylase/reductase: The ethylmalonyl-CoA pathway. Proc Natl Acad Sci USA 104:10631–10636. 17. Berk H, Buckel W, Thauer RK, Frey PA (1996) Re-face stereospecificity at C4 of NAD(P) for alcohol dehydrogenase from Methanogenium organophilum and for (R)-2hydroxyglutarate dehydrogenase from Acidaminococcus fermentans as determined by 1H-NMR spectroscopy. FEBS Lett 399:92–94. 18. Biellmann JF, Eid P, Hirth C, Jiirnvall H (1980) Aspartate-beta-semialdehyde dehydrogenase from Escherichia coli. Purification and general properties. Eur J Biochem 104:53–58. 19. Esaki N, et al. (1989) Enzymatic in situ determination of stereospecificity of NADdependent dehydrogenases. J Biol Chem 264:9750 –9752. 20. Erb TJ, Re´tey J, Fuchs G, Alber BE (2008) Ethylmalonyl-CoA mutase from Rhodobacter sphaeroides defines a new subclade of coenzyme B12-dependent acyl-CoA mutases. J Biol Chem 283:32283–32293.


21. Shaw LL, Engel PC (1984) The purification and properties of ox liver short-chain acyl-CoA dehydrogenase. Biochem J 218:511–520. 22. Lundberg NN, Thorpe C (1993) Inactivation of short-chain acyl-coenzyme-A dehydrogenase from pig liver by 2-pentenoyl-coenzyme-A. Arch Biochem Biophys 305:454 – 459. 23. Simon EJ, Shemin D (1953) The preparation of S-succinyl coenzyme A. J Am Chem Soc 75:2520. 24. Stadtman ER (1957) Preparation and assay of acyl coenzyme A and other thiol esters; use of hydroxylamine. Methods Enzymol 3:931–946. 25. Decker K (1959) in Die Aktivierte Essigsaüre. Das Coenzym A und Seine Acylderivate im Stoffwechsel der Zelle, (Enke, Stuttgart, Germany), pp 84 – 89. 26. Pollock VV, Barber MJ (2001) Kinetic and mechanistic properties of biotin sulfoxide reductase. Biochemistry 40:1430 –1440. 27. Cooper TG, Tchen TT, Wood HG, Benedict CR (1968) The carboxylation of phosphoenolpyruvate and pyruvate. The active species of ‘‘CO2’’ utilized by phosphoenolpyruvate carboxykinase, carboxytransphosphorylase, and pyruvate carboxylase. J Biol Chem 243:3857–3863. 28. (30) Lehman TC, Thorpe C (1990) Alternate electron acceptors for medium-chain acyl-CoA dehydrogenase: Use of ferricenium salts. Biochemistry 29:10594 –10602.

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Fig. S1. Enzymatic analysis of the stereochemistry of acryloyl-CoA carboxylation by using methylmalonyl-CoA epimerase and (2R)-methylmalonyl-CoA mutase. Reductive carboxylation of acryloyl-CoA by Ccr yields methylmalonyl-CoA that is further transformed into succinyl-CoA. In case of (2S)-methylmalonyl-CoA as reaction product (alternative a), methylmalonyl-CoA epimerase and (2R)-methylmalonyl-CoA mutase are required. In case of (2R)-methylmalonyl-CoA as reaction product (alternative b), (2R)-methylmalonyl-CoA mutase alone is sufficient for the formation of succinyl-CoA. For R. sphaeroides Ccr, reductive carboxylation was shown to follow alternative a.


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Fig. S2. Topicity, stereochemistry and conformations of crotonyl-CoA and NADPH. (A) re face and si face at C2 and C3 of crotonyl-CoA. (B) Conformations of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) cofactor and stereochemistry of the hydrogen atoms at C4 of the nicotinamide ring.


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Fig. S3. 1H-NMR spectra of enzymatically synthesized and purified [2H]-(4R)-NADPH and [2H]-(4S)-NADPH. (A) Overlay of the 1H-NMR spectra of [2H]-(4S)-NADPH (black), [2H]-(4R)-NADPH (red) and a mixture of [2H]-(4S)- and [2H]-(4R)-NADPH (blue). (B) 1H-NMR spectra of the methylene protons at C4 of the nicotinamide ring in detail. For reference spectra and chemical shifts, see refs.erences 17–19.


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Fig. S4. Analysis of amino acid sequences and stereospecificity of enoyl(-thioester) reductases (ERs) and related proteins. Neighbor- joining tree of ERs and related proteins, listed in Table S2. The stereospecificity of proteins shown in bold type has been experimentally verified or suggested from crystal structures. The tree was constructed by using ClustalW as implemented in the BioEdit 7.0.9.0 software package and neighbor-joining algorithms as implemented in the Tree-ConW1.3b software package. The accession numbers for the proteins and the corresponding sequences are listed in Tables S3 and S4, respectively.


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Table S1. Enzymes in biological carboxylation reactions Enzyme Ribulose-1,5- bisphosphate carboxylase/oxygenase (RuBisCO) (1) Phosphoenolpyruvate carboxylase (2) Phosphoenolpyruvate carboxykinase (3) ⬙Methylketone carboxylases⬙ (4,5) ⬙2-Oxoacid carboxylases⬙ (6,7) ⬙Acyl-CoA carboxylases⬙ (8, 9)

Vitamin K-dependent carboxylase (10) Pyrrole-2-carboxylate decarboxylase (11) ⬙2-Oxoacid synthases⬙ (12,13)

2-Ketopropyl-CoM carboxylase (14) Phenylphosphate carboxylase (15) Crotonyl-CoA carboxylase/reductase (6)

Substrate

Source of carbon

Cofactor(s)

Metals

CO2

–

Mg2⫹

3-Phosphoglycerate

Phosphoenolpyruvate

HCO3⫺

–

Mg2⫹

Oxaloacetate

Phosphoenolpyruvate

CO2

ATP (GTP)

Mg2⫹

Oxaloacetate

Acetone

CO2

ATP*

Mn2⫹

Acetoacetate

CO2 (?) HCO3-

2 ATP§ ATP, biotin

Mg2⫹ Mg2⫹

HCO3-

ATP, biotin

Mg2⫹

CO2

Vitamin K

Mn2⫹

Benzoylacetate Oxaloacetate Oxalosuccinate Malonyl-CoA Methylmalonyl-CoA 3-Methylglutaconyl-CoA Isohexenyl-glutaconyl-CoA g-Carboxyglutamic acid

HCO3-

Organic acid

?

Ribulose-1,5-phosphate

Acetophenone Pyruvic acid 2-Oxoglutaric acid Acetyl-CoA Propionyl-CoA 3-Methylcrotonyl-CoA Geranoyl-CoA Glutamic acid resiudes of proteins Pyrrole Acetyl-CoA

CO2

Reduced ferredoxin, thiamine diphosphate

[FeS]-cluster

Propionyl-CoA Succinyl-CoA S-(2-methylpropionyl)-CoA S-2-(indol-3-yl)acetyl-CoA 2-Ketopropyl-CoM

CO2

NADPH, FAD⫹

–

Phenylphosphate

CO2

–

K⫹, Mn2⫹

Crotonyl-CoA, (Acryloyl-CoA)

CO2

NADPH

–

Product

Pyrrole-2-carboxylate Pyruvate 2-Oxobutyrate 2-Oxoglutarate 3-Methyl-2-oxobutanoate Indolepyruvate Acetoacetate 4-Hydroxybenzoate Ethylmalonyl-CoA, (methyl-malonyl-CoA)

?, A role of iron in catalysis cannot be clearly ruled out. *Hydrolyzed into AMP and 2 inorganic phosphates. §Hydrolyzed into 2 ADP and 2 inorganic phosphates.


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Table S2. Domain architecture and stereochemistry of various ER and alcohol dehydrogenases Conserved domains (NCBI database) and amino acid region

Stereochemistry of reduction

NAD(P)H

C3

C2

4R 4R 4R 4R† 4R 4R 4R 4R 4R* 4R* 4R* 4R* 4R§ 4R§ nd nd

re re re re – – – – nd nd nd nd nd nd nd nd

si si re re – – – – nd nd nd nd nd nd nd nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

4S 4S 4S

si si –

si re –

4S 4S*

– –

– –

nd nd 4S

nd nd si

nd nd si

4S

si

si?

4S nd nd

– nd nd

– nd nd

Protein(fragment) and organism ER domain (FAS I) rat liver ER domain (FAS I) chicken Ccr Rhodobacter sphaeroides Ccr Streptomyces collinus AOR Rattus norvegicus Adh horse liver Adh Saccharomyces cervisiae Adh Pseudomonas fluorescens Qor Escherichia coli Qor Thermus thermophilus HB8 ␨-Crystallin human lens Etr1p Candida tropicalis Ccr Methylobacterium extorquens Ccr Streptomyces coelicolor A3(2) LovC Aspergillus terreus NysC ER domain (PKS I) Streptomyces noursei NysJ ER domain (PKS I) Streptomyces noursei EpoA ER domain (PKS I) Sorangium cellulosum EpoD ER domain 1 (PKS I) Sorangium cellulosum EpoD ER domain 2 (PKS I) Sorangium cellulosum ChcA Streptomyces collinus FabI (FAS II) Escherichia coli K12 InhA (FAS II) Mycobacterim tuberculosis Adh Drosophila melanogaster Sco1815 (PKS II) Streptomyces coelicolor A3(2) FabV Vibrio cholerae FabL Bacillus subtilis ER domain (FAS I) Saccharomyces cerevisiae ER domain (FAS I) Breibacterium ammoniagenes 2,4-DCR Escherichia coli K12 FabK Streptococcus pneumoniae Rv2953 Mycobacterium tuberculosis

ADH㛭zinc㛭N superfamily pfam00107

AdoHcyase superfamily cl09931

TIM㛭phosphate-binding super-family cl09108

50–200 50–185 190–320 225–380 165–290 210–375 190–310 170–285 140–280 190–245 170–310 170–300 210–375 225–385 165–260 110–235 110–250 115–260 80–220 115–250 15–255 1–262 1–269 1–190 20–240 1–401 1–250 10–285 100–250 1–360 10–230

nd, not determined. *Suggested from crystal structure. †Mistakenly described as 4S in the original publication (see text). §Suggested because of very high sequence similarity to Ccr of R. sphaeroides.


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Table S3. Accession numbers of the proteins analyzed in this study Accession number 1YB5 1PS9 1MG5 1HLD Q4KFF8 P00330 NP㛭620218.1 ZP㛭02056035.1 YP㛭354044.1 NP㛭630556.1 AAA92890.1 AAC44655.1 AAF62880.1 AAF62883.1 Q8WZM3.1 P0AEK4.2 AAF98273.1 NP㛭388745.1 ABX38717.1 CAA46024.1 P12276.5 P12785.3 NP㛭012739.1 CAB02034.1 3B6Z AAF71776.1 AAF71767.1 1QOR 1IYZ P95139.1 2NMO

Name

Function

– 2,4-DCR Adh Adh Adh Adh Aor

z-Crystallin 2,4-Dienoyl-CoA reductase Alcohol dehydrogenase Alcohol dehydrogenase Alcohol dehydrogenase Alcohol dehydrogenase Alkenal/alkenone oxidoreductase (Leukotriene B4 12-hydroxydehydrogenase) Crotonyl-CoA carboxylase/reductase Crotonyl-CoA carboxylase/reductase Crotonyl-CoA carboxylase/reductase Crotonyl-CoA reductase (carboxylase?) 1-Cyclohexenylcarbonyl CoA reductase Epothilone polyketide synthase module 0* Epothilone polyketide synthase module 3, 4, 5 and 6§ Trans-2 enoyl-(acyl-carrier-protein) reductase 1 (mitochondrial) Enoyl-(acyl-carrier-protein) reductase Trans-2-enoyl-(acyl-carrier-protein) reductase II Enoyl-(acyl carrier protein) reductase Enoyl-(acyl-carrier-protein) reductase IV Fatty acid synthase* Fatty acid synthase* Fatty acid synthase* Fatty acid synthase, b-subunit* Enoyl-(acyl-carrier-protein) reductase Lovastatin polyketide enoyl reductase Nystatin polyketide synthase, module 5 (steps 3–8)* Nystatin polyketide synthase, module 15 (steps 15–17)* Quinone oxidoreductase Quinone oxidoreductase Trans-acting enoyl reductase b-Ketoacyl-(acyl-carrier-protein) reductase

Ccr Ccr Ccr Ccr ChcA EpoA EpoD Etr1p FabI FabK FabL FabV FAS FAS FAS FAS InhA LovC NysC NysJ Qor Qor Rv2953 Sco1815

Organism Homo sapiens (lens) Escherichia coli Drosophila melanogaster Equus caballus (liver) Pseudomonas fluorescens Saccharomyces cerevisiae Rattus norvegicus Methylobacterium extorquens AM1 Rhodobacter sphaeroides 2.4.1. Streptomyces coelicolor A3(2) Streptomyces collinus Streptomyces collinus Sorangium (Polyangium) cellulosum Sorangium (Polyangium) cellulosum Candida tropicalis Escherichia coli Streptococcus pneumoniae Bacillus subtilis Vibrio cholerae Brevibacterium (Corynebacterium) ammoniagenes Gallus gallus Rattus norvegicus (liver) Saccharomyces cerevisiae Mycobacterium tuberculosis H37v Aspergillus terreus Streptomyces noursei Streptomyces noursei Escherichia coli Thermus thermophilus HB8 Mycobacterium tuberculosis H37v Streptomyces coelicolor A3(2)

*Contains 1 enoyl-thioester reductase domain that was identified and selected for amino acid sequence analysis. §Contains 2 enoyl-CoA reductase domains that were both identified and selected for amino acid sequence analysis.


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Table S4. Amino acid sequences of the proteins analyzed in this study (FASTA format) ⬎zeta-CRYSTALLIN㛭HOMO MHHHHHHSSGVDLGTENLYFQSMATGQKLMRAVRVFEFGGPEVLKLRSDIAVPIPKDHQVLIKVHACGVNPVETYIRSGTYSRKPLLPYTPGSDVAGVIEAVGDNASAFK KGDRVFTSSTISGGYAEYALAADHTVYKLPEKLDFKQGAAIGIPYFTAYRALIHSACVKAGESVLVHGASGGVGLAACQIARAYGLKILGTAGTEEGQKIVLQNGAHEVFN HREVNYIDKIKKYVGEKGIDIIIEMLANVNLSKDLSLLSHGGRVIVVGSRGTIEINPRDTMAKESSIIGVTLFSSTKEEFQQYAAALQAGMEIGWLKPVIGSQYPLEKVAEAHE NIIHGSGATGKMILLL ⬎2,4-DCR㛭ESCHERICHIA SYPSLFAPLDLGFTTLKNRVLMGSMHTGLEEYPDGAERLAAFYAERARHGVALIVSGGIAPDLTGVGMEGGAMLNDASQIPHHRTITEAVHQEGGKIALQILHTGRYSYQP HLVAPSALQAPINRFVPHELSHEEILQLIDNFARCAQLAREAGYDGVEVMGSEGYLINEFLTLRTNQRSDQWGGDYRNRMRFAVEVVRAVRERVGNDFIIIYRLSMLDLVE DGGTFAETVELAQAIEAAGATIINTGIGWHEARIPTIATPVPRGAFSWVTRKLKGHVSLPLVTTNRINDPQVADDILSRGDADMVSMARPFLADAELLSKAQSGRADEINTCI GCNQACLDQIFVGKVTSCLVNPRACHETKMPILPAVQKKNLAVVGAGPAGLAFAINAAARGHQVTLFDAHSEIGGQFNIAKQIPGKEEFYETLRYYRRMIEVTGVTLKLN HTVTADQLQAFDETILASGIVPRTPPIDGIDHPKVLSYLDVLRDKAPVGNKVAIIGCGGIGFDTAMYLSQPGESTSQNIAGFCNEWGIDSSLQQAGGLSPQGMQIPRSPRQIV MLQRKASKPGQGLGKTTGWIHRTTLLSRGVKMIPGVSYQKIDDDGLHVVINGETQVLAVDNVVICAGQEPNRALAQPLIDSGKTVHLIGGCDVAMELDARRAIAQGTRL ALEI ⬎ADH㛭DROSOPHILA SFTLTNKNVIFVAGLGGIGLDTSKELLKRDLKNLVILDRIENPAAIAELKAINPKVTVTFYPYDVTVPIAETTKLLKTIFAQLKTVDVLINGAGILDDHQIERTIAVNYTGLVNT TTAILDFWDKRKGGPGGIICNIGSVTGFNAIYQVPVYSGTKAAVVNFTSSLAKLAPITGVTAYTVNPGITRTTLVHKFNSWLDVEPQVAEKLLAHPTQPSLACAENFVKAIEL NQNGAIWKLDLGTLEAIQWTKHWDSGI ⬎ADH㛭EQUUS STAGKVIKCKAAVLWEEKKPFSIEEVEVAPPKAHEVRIKMVATGICRSDDHVVSGTLVTPLPVIAGHEAAGIVESIGEGVTTVRPGDKVIPLFTPQCGKCRVCKHPEGNFCL KNDLSMPRGTMQDGTSRFTCRGKPIHHFLGTSTFSQYTVVDEISVAKIDAASPLEKVCLIGCGFSTGYGSAVKVAKVTQGSTCAVFGLGGVGLSVIMGCKAAGAARIIGVD INKDKFAKAKEVGATECVNPQDYKKPIQEVLTEMSNGGVDFSFEVIGRLDTMVTALSCCQEAYGVSVIVGVPPDSQNLSMNPMLLLSGRTWKGAIFGGFKSKDSVPKLVA DFMAKKFALDPLITHVLPFEKINEGFDLLRSGESIRTILTF ⬎ADH㛭PSEUDOMONAS MPQTLTLNQRVVLVSRPEGAPVPENFRLERVALPELADGQVLLKTLYLSLDPYMRGRMSDAPSYAAPVEIDEVMTGGAVSRVERSLNPKFQEGDLVVGATGWQSHCICD GRNLIPVPSGLPSPSMALGVLGMPGMTAYMGLMDIGQPKAGETLVVGAASGAVGSVVGQVAKLKGLRVVGVAGGADKCRYVVEELGFDACIDHKSPDFADELAQACF KGVDIYFENVGGKVFDGVLPLLNPRARIPLCGLIAQYNAQALPPGPDRLPLLQRTLLTKRVRIQGFIVFDDYGDRHPEFIKAMAPWVREGKVKFKEDVVEGLEQAPEAFIGL LEGRNFGKLVVKVAPDASI ⬎ADH㛭SACCHAROMYCES MSIPETQKGVIFYESHGKLEHKDIPVPKPKANELLINVKYSGVCHTDLHAWHGDWPLPVKLPLVGGHEGAGVVVGMGENVKGWKIGDYAGIKWLNGSCMACEYCELGN ESNCPHADLSGYTHDGSFQQYATADAVQAAHIPQGTDLAQVAPILCAGITVYKALKSANLMAGHWVAISGAAGGLGSLAVQYAKAMGYRVLGIDGGEGKEELFRSIGGE VFIDFTKEKDIVGAVLKATDGGAHGVINVSVSEAAIEASTRYVRANGTTVLVGMPAGAKCCSDVFNQVVKSISIVGSYVGNRADTREALDFFARGLVKSPIKVVGLSTLPEI YEKMEKGQIVGRYVVDTSK ⬎AOR㛭RatTUS MVQAKTWTLKKHFEGFPTDSNFELRTTELPPLNNGEVLLEALFLSVDPYMRVAAKKLKEGDSMMGEQVARVVESKNSAFPTGTIVVALLGWTSHSISDGNGLRKLPAEW PDKLPLSLALGTVGMPGLTAYFGLLDICGLKGGETVLVNAAAGAVGSVVGQIAKLKGCKVVGTAGSDEKVAYLKKLGFDVAFNYKTVKSLEEALRTASPDGYDCYFDN VGGEFSNTVILQMKTFGRIAICGAISQYNRTGPCPPGPSPEVIIYQQLRMEGFIVTRWQGEVRQKALTDLMNWVSEGKIRYHEYITEGFEKMPAAFMGMLKGDNLGKTIVKA ⬎CCR㛭methylobacterium MAASAAPAWTGQTAEAKDLYELGEIPPLGHVPAKMYAWAIRRERHGPPEQSHQLEVLPVWEIGDDEVLVYVMAAGVNYNGVWAGLGEPISPFDVHKGEYHIAGSDASG IVWKVGAKVKRWKVGDEVIVHCNQDDGDDEECNGGDPMFSPTQRIWGYETGDGSFAQFCRVQSRQLMARPKHLTWEEAACYTLTLATAYRMLFGHAPHTVRPGQNV LIWGASGGLGVFGVQLCAASGANAIAVISDESKRDYVMSLGAKGVINRKDFDCWGQLPTVNSPEYNTWLKEARKFGKAIWDITGKGNDVDIVFEHPGEATFPVSTLVAK RGGMIVFCAGTTGFNITFDARYVWMRQKRIQGSHFAHLKQASAANQFVMDRRVDPCMSEVFPWDKIPAAHTKMWKNQHPPGNMAVLVNSTRAGLRTVEDVIEAGPLKAM ⬎CCR㛭Rhodobacter MALDVQSDIVAYDAPKKDLYEIGEMPPLGHVPKEMYAWAIRRERHGEPDQAMQIEVVETPSIDSHEVLVLVMAAGVNYNGIWAGLGVPVSPFDGHKQPYHIAGSDASGI VWAVGDKVKRWKVGDEVVIHCNQDDGDDEECNGGDPMFSPTQRIWGYETPDGSFAQFTRVQAQQLMKRPKHLTWEEAACYTLTLATAYRMLFGHKPHDLKPGQNVL VWGASGGLGSYAIQLINTAGANAIGVISEEDKRDFVMGLGAKGVINRKDFKCWGQLPKVNSPEYNEWLKEARKFGKAIWDITGKGINVDMVFEHPGEATFPVSSLVVKK GGMVVICAGTTGFNCTFDVRYMWMHQKRLQGSHFANLKQASAANQLMIERRLDPCMSEVFPWAEIPAAHTKMYRNQHKPGNMAVLVQAPRTGLRTFADVLEAGRKA ⬎CCR㛭streptomyces㛭Coelicolor MTVKDILDAIQSPDSTPADIAALPLPESYRAITVHKDETEMFAGLETRDKDPRKSIHLDDVPVPELGPGEALVAVMASSVNYNSVWTSIFEPLSTFGFLERYGRVSDLAKRH DLPYHVIGSDLAGVVLRTGPGVNAWQAGDEVVAHCLSVELESSDGHNDTMLDPEQRIWGFETNFGGLAEIALVKSNQLMPKPDHLSWEEAAAPGLVNSTAYRQLVSRN GAGMKQGDNVLIWGASGGLGSYATQFALAGGANPICVVSSPQKAEICRAMGAEAIIDRNAEGYRFWKDENTQDPKEWKRFGKRIRELTGGEDIDIVFEHPGRETFGASVF VTRKGGTITTCASTSGYMHEYDNRYLWMSLKRIIGSHFANYREAWEANRLIAKGRIHPTLSKVYSLEDTGQAAYDVHRNLHQGKVGVLCLAPEEGLGVRDREKRAQHLD AINRFRNI ⬎CCR㛭streptomyces㛭collinus MTVKDILDAIQSKDATSADFAALQLPESYRAITVHKDETEMFAGLETRDKDPRKSIHLDEVPVPELGPGEALVAVMASSVNYNSVWTSIFEPVSTFAFLERYGKLSPLTKRH DLPYHIIGSDLAGVVLRTGPGVNAWQPGDEVVAHCLSVELESPDGHDDTMLDPEQRIWGFETNFGGLAEIALVKTNQLMPKPKHLTWEEAAAPGLVNSTAYRQLVSRNG AAMKQGDNVLIWGASGGLGSYATQFALAGGANPICVVSSPQKAEICRSMGAEAIIDRNAEGYKFWKDEHTQDPKEWKRFGKRIRELTGGEDIDIVFEHPGRETFGASVYV TRKGGTITTCASTSGYMHEYDNRYLWMSLKRIIGSHFANYREAYEANRLIAKGKIHPTLSKTYSLEETGQAAYDVHRNLHQGKVGVLCLAPEEGLGVRDAEMRAQHIDAI NRFRNV ⬎ChcA㛭STREPTOMYCES㛭collinus MNSPHQQQTADRRQVSLITGASRGIGRTLALTLARRGGTVVVNYKKNADLAQKTVAEVEEAGGQGFAVQADVETTEGVTALFDEVAQRCGRLDHFVSNAAASAFKNIV DLGPHHLDRSYAMNLRPFVLGAQQAVKLMDNGGRIVALSSYGSVRAYPTYAMLGGMKAAIESWVRYMAVEFAPYGINVNAVNGGLIDSDSLEFFYNVEGMPPMQGVL DRIPARRPGTVQEMADTIAFLLGDGAGYITGQTLVVDGGLSIVAPPFFADAGEALELPPRPTRDA ⬎EpoA㛭Fragment㛭Sorangium PGLGEVEIAVDAAGLSFNDVQLALGMVPDDLPGKPNPPLLLGGECAGRIVAVGEGVNGLVVGQPVIALSAGAFATHVTTSAALVLPRPQALSATEAAAMPVAYLTAWYA LDGIARLQPGERVLIHAATGGVGLAAVQWAQHVGAEVHATAGTPEKRAYLESLGVRYVSDSRSDRFVADVRAWTGGEGVDVVLNSLSGELIDKSFNLLRSHGRFVELGK RDCYADNQLGLRPFLRNLSFSLVDLRGMMLERPARVRALFEELLGLIAAGVFTPPPIATLPIARVADAFRSMAQAQHLGKLVLTLGDPEVQIRIPTHAGAGP


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⬎epod㛭Fragment㛭1㛭sorangium AGPMGGDCAGIVTAVGQGVHHLSVGDAVMTLGTLHRFVTVDARLVVRQPAGLTPAQAATVPVAFLTAWLALHDLGNLRRGERVLIHAAAGGVGMAAVQIARWIGAEV FATASPSKWAAVQAMGVPRTHIASSRTLEFAETFRQVTGGRGVDVVLNALAGEFVDASLSLLTTGGRFLEMGKTDIRDRAAVAAAHPGVRYRVFDILELAPDRTREILER VVEGFAAGHLRALPVHAFAITKAEAAFRFMAQARHQGKVVLLPAPSAAPLAPTGTVLLTGGLGALGLHVARWLAQQGAPHMVL ⬎epod㛭fragment㛭2㛭Sorangium RRPPGPGEVEIAVEAAGLNFLDVMRAMGIYPGPGDGPVALGAECSGRIVAMGEGVESLRIGQDVVAVAPFSFGTHVTIDARMVAPRPAALTAAQAAALPVAFMTAWYG LVHLGRLRAGERVLIHSATGGTGLAAVQIARHLGAEIFATAGTPEKRAWLREQGIAHVMDSRSLDFAEQVLAATKGEGVDVVLNSLSGAAIDASLATLVPDGRFIELGKTD IYADRSLGLAHFRKSLSYSAVDLAGLAVRRPERVAALLAEVVDLLARGALQPLPVEIFPLSRAADAFRKMAQAQHLGKLVLALEDPDVRIRVPGESGVAIRADGTYLVTGG LGGLGLSVAGWLAEQGAGHLVLVG ⬎Etr1p㛭CANDIDA MITAQAVLYTQHGEPKDVLFTQSFEIDDDNLAPNEVIVKTLGSPVNPSDINQIQGVYPSKPAKTTGFGTTEPAAPCGNEGLFEVIKVGSNVSSLEAGDWVIPSHVNFGTWRT HALGNDDDFIKLPNPAQSKANGKPNGLTINQGATISVNPLTAYLMLTHYVKLTPGKDWFIQNGGTSAVGKYASQIGKLLNFNSISVIRDRPNLDEVVASLKELGATQVITED QNNSREFGPTIKEWIKQSGGEAKLALNCVGGKSSTGIARKLNNNGLMLTYGGMSFQPVTIPTSLYIFKNFTSAGFWVTELLKNNKELKTSTLNQIIAWYEEGKLTDAKSIET LYDGTKPLHELYQDGVANSKDGKQLITY ⬎FabI㛭ESCHERICHIA MGFLSGKRILVTGVASKLSIAYGIAQAMHREGAELAFTYQNDKLKGRVEEFAAQLGSDIVLQCDVAEDASIDTMFAELGKVWPKFDGFVHSIGFAPGDQLDGDYVNAVT REGFKIAHDISSYSFVAMAKACRSMLNPGSALLTLSYLGAERAIPNYNVMGLAKASLEANVRYMANAMGPEGVRVNAISAGPIRTLAASGIKDFRKMLAHCEAVTPIRRT VTIEDVGNSAAFLCSDLSAGISGEVVHVDGGFSIAAMNELELK ⬎fabK㛭STREPTOCOCCUS MKTRITELLKIDYPIFQGGMAWVADGDLAGAVSKAGGLGIIGGGNAPKEVVKANIDKIKSLTDKPFGVNIMLLSPFVEDIVDLVIEEGVKVVTTGAGNPSKYMERFHEAGII VIPVVPSVALAKRMEKIGADAVIAEGMEAGGHIGKLTTMTLVRQVATAISIPVIAAGGIADGEGAAAGFMLGAEAVQVGTRFVVAKESNAHPNYKEKILKARDIDTTISAQ HFGHAVRAIKNQLTRDFELAEKDAFKQEDPDLEIFEQMGAGALAKAVVHGDVDGGSVMAGQIAGLVSKEETAEEILKDLYYGAAKKIQEEASRWTGVVRND ⬎fabL㛭BACILLUS MEQNKCALVTGSSRGVGKAAAIRLAENGYNIVINYARSKKAALETAEEIEKLGVKVLVVKANVGQPAKIKEMFQQIDETFGRLDVFVNNAASGVLRPVMELEETHWDWT MNINAKALLFCAQEAAKLMEKNGGGHIVSISSLGSIRYLENYTTVGVSKAALEALTRYLAVELSPKQIIVNAVSGGAIDTDALKHFPNREDLLEDARQNTPAGRMVEIKDM VDTVEFLVSSKADMIRGQTIIVDGGRSLLV ⬎fabV㛭VIBRIO MIIKPKIRGFICTTTHPVGCEANVKEQIAYTKAQGPIKNAPKRVLVVGSSSGYGLSSRIAAAFGGGAATIGVFFEKPGTDKKPGTAGFYNAAAFDKLAHEAGLYAKSLNGDA FSNEAKQKAIELIKQDLGQIDLVVYSLASPVRKMPDTGELVRSALKPIGETYTSTAVDTNKDVIIEASVEPATEQEIADTVTVMGGQDWELWIQALEEAGVLAEGCKTVAY SYIGTELTWPIYWDGALGRAKMDLDRAATALNEKLAAKGGTANVAVLKSVVTQASSAIPVMPLYIAMVFKKMREQGVHEGCMEQIYRMFSQRLYKEDGSAPEVDDHN RLRLDDWELRDDIQQHCRDLWPQITTENLRELTDYDMYKEEFIKLFGFGIEGIDYDADVNPEVEFDVIDIE ⬎FAS㛭BREVIbacterium TRFTELTGYSPVVLAGMTPSTVDPAIVAAAANAGFWAELAGGGQVTDAILNDSLERLEDMLNPGINAQFNAMYLSPKQWRAQIEGRRLIPRARANGASINGVICSAGIPPH EEIALVRQLQEDNIPWVAFKPGAVRHVHQVLAIADDLPDTTVIMQVEGGKAGGHHSWEDLSSLLTETYADIRERDNVVLMAAGGIGAPERGAQYLTGEWSKVYGLPAMP VDAIMIGTAAMATKESTASESVKQALVATQGLEDIPGGGWVPAGGARDGIASGRSQLGADIHEIDNTFAKAGRLLDE ⬎FAS㛭FRAGMENT㛭GALLUS PAKGLATVVDCDKRFLWEVPENWTLEEAASVPVVYATAYYALVVRGGMKKGESVLIHSGSGGVGQAAIAIALSMGCRVFATVGSAEKREYLQARFPQLDANSFASSRNT TFQQHILRVTNGKGVSLVLNSLAEEKLQASLRCLAQHGRFLEIGKFDLSNNSQLGMALFLKNVAFHGILLDSIFEEGNQEWEVVSELLTKGIKDGVVKPLRTTVFGKEEVEA AFRFMAQGKHIGKVMIKIQEEEKQYPLRSEPVKLSAIS ⬎FAS㛭FRAGMENT㛭RATTUS VPAEGLATSVLLSPDFLWDVPSSWTLEEAASVPVVYTTAYYSLVVRGRIQHGETVLIHSGSGGVGQAAISIALSLGCRVFTTVGSAEKRAYLQARFPQLDDTSFANSRDTSF EQHVLLHTGGKGVDLVLNSLAEEKLQASVRCLAQHGRFLEIGKFDLSNNHPLGMAIFLKNVTFHGILLDALFEGANDSWREVAELLKAGIRDGVVKPLKCTVFPKAQVED AFRYMAQGKHIGKVLVQVREEEPEAMLPGAQPTLISAI ⬎FAS㛭FRAGMENT㛭SACCHAROMYCES TKFSKLIGRPPLLVPGMTPCTVSPDFVAATTNAGYTIELAGGGYFSAAGMTAAIDSVVSQIEKGSTFGINLIYVNPFMLQWGIPLIKELRSKGYPIQFLTIGAGVPSLEVASEYI ETLGLKYLGLKPGSIDAISQVINIAKAHPNFPIALQWTGGRGGGHHSFEDAHTPMLQMYSKIRRHPNIMLIFGSGFGSADDTYPYLTGEWSTKFDYPPMPFDGFLFGSRVMI AKEVKTSPDAKKCIAACTGVPDDKWEQTYKKPTGGIVTVRSEMGEPIHKIATRGVMLWKEFDE ⬎inhA㛭MYCOBACTERIUm MTGLLDGKRILVSGIITDSSIAFHIARVAQEQGAQLVLTGFDRLRLIQRITDRLPAKAPLLELDVQNEEHLASLAGRVTEAIGAGNKLDGVVHSIGFMPQTGMGINPFFDAPY ADVSKGIHISAYSYASMAKALLPIMNPGGSIVGMDFDPSRAMPAYNWMTVAKSALESVNRFVAREAGKYGVRSNLVAAGPIRTLAMSAIVGGALGEEAGAQIQLLEEGW DQRAPIGWNMKDATPVAKTVCALLSDWLPATTGDIIYADGGAHTQLL ⬎lovc㛭Aspergillus MGDQPFIPPPQQTALTVNDHDEVTVWNAAPCPMLPRDQVYVRVEAVAINPSDTKMRGQFATPWAFLGTDYAGTVVAVGSDVTHIQVGDRVYGAQNEMCPRTPDQGAF SQYTVTRGRVWAKIPKGLSFEQAAALPAGISTAGLAMKLLGLPLPSPSADQPPTHSKPVYVLVYGGSTATATVTMQMLRLSGYIPIATCSPHNFDLAKSRGAEEVFDYRAP NLAQTIRTYTKNNLRYALDCITNVESTTFCFAAIGRAGGHYVSLNPFPEHAATRKMVTTDWTLGPTIFGEGSTWPAPYGRPGSEEERQFGEDLWRIAGQLVEDGRLVHHPL RVVQGGFDHIKQGMELVRKGELSGEKLVVRLEGPLEHHHHHH ⬎NYSC㛭FRAGMEnt㛭StreptomyceS㛭Noursei LTGHEVRVEVRAAGLNFRDVLNALGMYPGDDVGSFGSEAAGVVVEVGPEVTGLAPGDQVMGMITGSFGSLAVDDARRLARLPEDWSWETGASVPLVFLTAYYALKEL GGLRAGEKVLVHAGAGGVGMAAIQIARHVGAEVFATASEGKWDVLRSLGVADDHIASSRTLDFEAAFAEVAGDRGLDVVLNSLAGDFVDASMRLLGDGGRFLEMGKT DIRAADSVPDGLSYQSFDLAWVVPETIGTMLAELMDLFRTGALRPLPVRTWDVRHAKDAFRFMSMAKHIGKIVLTL ⬎nysJ㛭Fragment㛭streptomyces㛭noursei RRPLTGHEVRVGIRAAGLNFRDVLNALGMYPGDAGLFGSEAAGVVVEVGPEVTGLAPGDRVMGMLFGGFGPLGIADARLLTPVPADWSWETGASVPLVFLTAYYALKE LGGLRAGEKVLVHAGAGGVGMAAIQIARHVGAEVFATASEGKWDVLRSLGVADDHIASSRTLDFEAAFAEVAGDRGLDVVLNALSGEFVDASMRLLGDGGRFLEMGKT DIRAADSVPDGLSYHSFDLGMVDPEHIQRMLLDLVELFDRGALAALPVRSWDVRRAGEAFRFMSLAQHIGKIVLTVPQPLDPDG ⬎qor㛭ESCHERICHIA MATRIEFHKHGGPEVLQAVEFTPADPAENEIQVENKAIGINFIDTYIRSGLYPPPSLPSGLGTEAAGIVSKVGSGVKHIKAGDRVVYAQSALGAYSSVHNIIADKAAILPAAIS FEQAAASFLKGLTVYYLLRKTYEIKPDEQFLFHAAAGGVGLIACQWAKALGAKLIGTVGTAQKAQSALKAGAWQVINYREEDLVERLKEITGGKKVRVVYDSVGRDTWE


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RSLDCLQRRGLMVSFGNSSGAVTGVNLGILNQKGSLYVTRPSLQGYITTREELTEASNELFSLIASGVIKVDVAEQQKYPLKDAQRAHEILESRATQGSSLLIP ⬎QOR㛭THERMUS MKAWVLKRLGGPLELVDLPEPEAEEGEVVLRVEAVGLNFADHLMRLGAYLTRLHPPFIPGMEVVGVVEGRRYAALVPQGGLAERVAVPKGALLPLPEGLSPEEAAAFPV SFLTAYLALKRAQARPGEKVLVQAAAGALGTAAVQVARAMGLRVLAAASRPEKLALPLALGAEEAATYAEVPERAKAWGGLDLVLEVRGKEVEESLGLLAHGGRLVYI GAAEGEVAPIPPLRLMRRNLAVLGFWLTPLLREGALVEEALGFLLPRLGRELRPVVGPVFPFAEAEAAFRALLDRGHTGKVVVRL ⬎rv2953㛭mycobacterium MSPAEREFDIVLYGATGFSGKLTAEHLAHSGSTARIALAGRSSERLRGVRMMLGPNAADWPLILADASQPLTLEAMAARAQVVLTTVGPYTRYGLPLVAACAKAGTDYA DLTGELMFCRNSIDLYHKQAADTGARIILACGFDSIPSDLNVYQLYRRSVEDGTGELCDTDLVLRSFSQRWVSGGSVATYSEAMRTASSDPEARRLVTDPYTLTTDRGAEP ELGAQPDFLRRPGRDLAPELAGFWTGGFVQAPFNTRIVRRSNALQEWAYGRRFRYSETMSLGKSMAAPILAAAVTGTVAGTIGLGNKYFDRLPRRLVERVTPKPGTGPSR KTQERGHYTFETYTTTTTGARYRATFAHNVDAYKSTAVLLAQSGLALALDRDRLAELRGVLTPAAAMGDALLARLPGAGVVMGTTRLS ⬎SCO1815㛭STREPTOMYCES㛭COELICOLOR MGSSHHHHHHSSGLVPRSHMSRSVLVTGGNRGIGLAIARAFADAGDKVAITYRSGEPPEGFLAVKCDITDTEQVEQAYKEIEETHGPVEVLIANAGVTKDQLLMRMSEED FTSVVETNLTGTFRVVKRANRAMLRAKKGRVVLISSVVGLLGSAGQANYAASKAGLVGFARSLARELGSRNITFNVVAPGFVDTDMTKVLTDEQRANIVSQVPLGRYAR PEEIAATVRFLASDDASYITGAVIPVDGGLGMGH


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