Identification, Expression, and Deduced Primary Structure of ...

Vol ,266, No.

THEJOURNAL OF BIOLOGICAL CHEMISTRY

0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Issue of October 25, pp. 20447-20452,1991 Printed in U.S. A .

Identification, Expression, and Deduced Primary Structure of Transketolase and Other Enzymes Encoded Within the Form I1 COa Fixation Operon ofRhodobacter sphaeroides” (Received for publication, March 14, 1991)

Jiann-Hwa ChenS, Janet L. Gibson§, Lee Ann McCueg, and F. Robert Tabitatll From the $Department of Microbiology and Center for Applied Microbiology, The University of Texas, Austin, Texas 78712 and the §Department of Microbiology and The Biotechnology Center, The Ohio State University, Columbus, Ohio 43210

Previous studies had indicated that the form I1 or B carboxylase, consisting of both large (catalytic)and small cluster of COz fixation structural genes is part of a subunits, resembles the plant enzyme in its structure and large operon in Rhodobacter sphaeroides (Gibson, J. catalytic properties, while the form I1 enzyme consists only L., Chen, J.-H., Tower, P. A., and Tabita,F. R. (1990) of large subunits, much like the enzyme from Rhodospirillum Biochemistry 29, 8086-8093). In this investigation, rubrum (Tabita and McFadden, 1974; Nargang et al., 1984; we have sequenced the DNA between the prkB and Gibson and Tabita, 1977; Jordan and Ogren, 1981; Wagner et rbpL genesandprovideevidenceforthreedistinct al., 1988). More recently distinct Rbu-P2 carboxylase genes, open reading frames which encode additional strucrbcL rbcS and rbpL, coding for the form I and form I1Rbu-Ps tural genes of the Calvin reductive pentose phosphate carboxylase subunit polypeptides, respectively, were found on pathway; these genes encode the enzymes transketolase, glyceraldehyde phosphate dehydrogenase, and al- the R. sphueroides chromosome (Quivey and Tabita, 1984; Muller et al., 1985; Gibson andTabita, 1986; Rainey and dolase.Noteworthy is transketolase,which maybe expressed to high levels in Escherichia coli.This study Tabita, 1989). It was subsequently found that the form I and thus represents the initial description of the primary form I1 Rbu-Pz carboxylase genes are located downstream structure of bacterial transketolase, a key enzyme of from a cluster of additional Cos fixation structural genes, the reductiveandthe oxidative pentosephosphate including genes encoding phosphoribulokinase ( p r k ) (Gibson pathways. Each of the genes are separated by short and Tabita, 1987; Hallenbeck andKaplan, 1987), fructose stretches of intergenic sequence, consistent with ear- bisphosphatase ( f b p )(Gibson and Tabita, 1988),and proteins lier evidencewhichsuggestedthat these genes are of unknown function, the products of the cfx genes (Hallencotranscribed and part oflarge a operon controlledby beck andKaplan, 1987; Gibson andTabita, 1988). These sequences upstreamfrom fbpB. genes were found to be organized somewhat differently in the two clusters of the R. sphaeroides chromosome, however, both prkA/fbpA and prkB/fbpB were tightly associated in the A In most photosynthetic and chemoautotrophic organisms, and B clusters, respectively, with prkA and fbpA apparently COz is fixed into organic carbon by means of the reductive cotranscribed when expressed in Escherichia coli (Gibson and pentose phosphate pathway. This carbon assimilatory route Tabita, 1988). In addition, p r k B and fbpB were recently found is conserved throughout evolution, from bacteria to higher to be cotranscribed and are part of a large operon in R. plants. Due to their metabolic diversity andpotential for sphueroides (Gibson et al., 1990). The major difference in the elucidating underlying mechanisms of control, microorga- organization of the two pentose phosphate structural gene nisms have increasingly been employed for studies on CO, clusters was the large stretch of about 3000-nucleotide base fixation (Tabita, 1987; 1988), and the purple nonsulfur pho- pairs (Fig. 1) separating p r k B from cfnB in the B cluster. tosynthetic bacterium Rhodobacter sphaeroides has proven to Analogous sequences are not found between prkA and cfxA of be an especially interesting organism for such studies. For the A cluster (Gibson and Tabita, 1988). example, several years ago it was found that R. sphueroides The goal of this study was to analyze the unique 3000-base synthesizes two distinct structural forms of the key enzyme pair region of the B cluster to better understandthe molecular ribulose 1,5-bisphosphate carboxylase/oxygenase (Rbu-Pz organization, function, and regulation of the two clusters of carboxylase)’ (Gibson and Tabita, 1977). The form I Rbu-P2 COz fixation genes in R. sphaeroides.

* This work was supported by United States Department of Agriculture Grants 89-37262-4566 and 87-CRCR-1-2591. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solelyto indicate this fact. The nucleotide seqwnce(s)reported in thispaper has been submitted to the GenBankTM/EMBL Data Bank withaccessionnumber($ M68914. (1 To whom correspondence should be addressed Department of Microbiology, The Ohio State University, 484 West 12th Ave., Columbus, OH 43210. ‘The abbreviations used are: Rbu-P, carboxylase, ribulose bisphosphate carboxylase/oxygenase; IPTG, isopropyl P-D-thiogalactoside; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; kb, kilobase pairs; ORF, open reading frame.

EXPERIMENTALPROCEDURES

Plasmid Propagation and DNA Manipulation-Different regions of the 4-kb EcoRI fragment of plasmid pJGlO6, containing the CO, fixation structural genes of the B region of R. sphueroides,were cloned into pUC8 vectors (Vieira and Messing, 1982) as previously reported (Gibson and Tabita,1987; 1988). Luria broth (LB) (Davis et al., 1980) was used for bacterial cultivation, and ampicillin, at theconcentration of 50 pg/ml, was added to avoid plasmid segregation. DNA Sequencing-The sequencing strategy initially involved the generation of subclones containing various restriction fragments within, upstream, and downstream of the 4-kb EcoRI fragment of pJGlO6. Plasmid DNA of each subclone was then sequenced using both universal and reverse pUC8 sequencing primers. Generally, from one sequencing reaction, 300-400 nucleotides were read. For regions beyond this area, a 15-mer oligonucleotide containing the end se-

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Transketohe and Other Enzymes

20448

quence of the last reading was commercially synthesized (Operon Technologies, Inc., San Pablo, CA) and used as a sequencing primer forfurther sequencing. Sequencing was carried out on denatured double stranded DNA according to instructions of the "Sequenase" kit (United States Biochemical Corp., Cleveland, OH). For labeling, 13'S]dATP or [36S]dCTPwas used and purchased from Du Pont-New England Nuclear at a specific activity of 3000 Ci/mol. Both strands of the EcoRI fragment were sequenced. Because the nucleotide aequence is G-C rich, at least one strand was also sequenced using a dITP or deaza-GTP reaction mixture, as suggested in the Sequenase kit. This procedure resolved G-C compression. Growth of Cells, Preparation of Extrocts, and Enzyme Assays-R. sphueroides was grown photolithoautotrophically in an atmosphere of1.5% Cor, 98.5% H2 as previously described (Jouanneau and Tabita, 1986), and extracts were prepared from sonically disrupted cells (Gibson and Tabita,1987). Plasmid and nonplasmid containing E. coli JM107 were grown in L broth. Approximately 100 pl of an overnight culture was used to inoculate 100 ml of fresh medium in a 500-ml Erlenmeyer flask. After 1 h, isopropyl @-D-thiogalactoside (IPTG) was added, where required, to a final concentration of 1 mM. After 3-4 h, the cells were harvested, washed twice with cold buffer, 20 mM Tris-C1, pH 7.5, 1 mM EDTA, 10 mM @-mercaptoethanol (TEM buffer), and stored frozen at -70 "C. At the desired time, the cells were thawed in cold TEM andsonicated. Transketolase catalyzes the transfer of the two-carbon glycoaldehyde group of xylulose 5-phosphate to ribose 5-phosphate to generate glyceraldehyde 3-phosphate and sedoheptulose 7-phosphate. The enzyme was assayed by followingthe oxidation of NADH by dihydroxyacetone phosphate in the presence of a-glycerophosphate dehydrogenase/triose-phosphate isomerase. The assay contained 100 mM Tris-C1, pH 7.5, 20 mM xylulose 5-phosphate, 20 mM ribose 5phosphate, 50 p~ NADH, 1 mM thiamine pyrophosphate, 10 mM MgCI2.6H2O,2 units of a-glycerophosphate dehydrogenase, and 32 units of triose-phosphate isomerase, and extract to1 ml. A commercial preparation of yeast transketolase (Sigma) was used to verify that theassay procedure and reagents functioned properly. Glyceraldehyde-3phosphate dehydrogenase (GAPDH) activity was measured in a reaction mixture containing 100 mM Tris-C1, pH 8.5, 17 mM sodium arsenate, pH 8.5, 4 mM freshly neutralized L-cysteine hydrochloride, 20 mM sodium fluoride, 4 mM NAD', 8 mM DLglyceraldehyde 3-phosphoric acid, and extract to 1 ml. Both spectrophotometric assays were performed at 340 nm in a Beckman model 70 recording spectrophotometer at ambient temperature. Appropriate controls were runineach case so that only substrate-dependent linear activity rates were measured. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed as previously described (Gibson and Tabita, 1986); protein levels were determined by a modified Lowry procedure (Markwell et al., 1978). RESULTS AND DISCUSSION

Sequence Analysis of the Region Between prkB and rbpLThe form I1 CO, fixation cluster in R. sphueroides contains the previously identified genes, fbpB, prkB, gapB, cfxB, and rbpL (Fig. 1).The nucleotide sequences for fbpB and prkB (Gibson et al., 1990) and rbpL (Wagner et al., 1988) have been

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reported. In this study, we determined the sequence of a 4.1kb EcoRI fragment that contains the coding sequence for the entire putative gapB gene, a partial coding sequence for cfxB (Gibson and Tabita,1988; Hallenbeck and Kaplan, 1987),and 2 kb of DNA of unidentified function situated between prkB and gapB (Fig. 1). The complete nucleotide sequence and deduced amino acid sequences derived from this fragment are shown (Fig. 2). A large open reading frame (ORF) of 1974 nucleotides was found by computer analysis in the DNA sequence between prkB and gapB. This ORF begins at an ATG codon situated 80 nucleotides downstream from the translation stop siteof prkB and extends to a TGA codon at position 2161 (Fig. 2). The open reading frame would encode a polypeptide of 657 amino acids with a predicted molecular weight of 69,341. Homology searches of the entire known sequence bank (GenBank) yielded only one possible clue as to the identification of this gene. This is thegene that encodes the enzyme dihydroxyacetone synthase (accession number: A23009), a key methanol assimilating enzyme in the methylotrophic yeast Hansenula polymorph. Indeed dihydroxyacetone synthase is in reality a novel type of transketolase which catalyzes a very similar reaction to the conventional transketolase typical of the oxidative and reductive pentose phosphatepathways. This enzyme comprises 702 amino acid residues in H. polymorph (Janowicz et al., 1985). Dot plots comparing the deduced R. sphueroides amino acid sequence and the dihydroxyacetone synthase sequence of H. polymorph (Fig. 3) showed several regions of precise homology. Further alignment (Fig. 4) indicated that there was 35% identity and 56% similarity with the previously determined amino acid sequence of H. polym o r p h dihydroxyacetone synthase. Several large stretches are identical and may be of functional significance, such as potential nucleotide-binding sites at conserved regions between residues 105-115 and 446-463of the R. sphueroides sequence (Fig. 5). These resultsstrongly indicate that this R. sphueroides ORF may encode a transketolase (tklB) gene. Given the importance of this enzyme in pentose metabolism in general, and in the COz assimilatory pathway in particular, it was not surprisingto find thisgene in a cluster of structural genes encoding enzymes of COz fixation. Nucleotide and Amino Acid Sequence of the Putative Gap Sequence-Ten nucleotides beyond the stop codon of the transketolase ORF is the initiation codon for another ORF encoding 334 amino acids from nucleotide 3986 to nucleotide 4987 (Fig. 2). On the basis of Southern blotswith heterologous probes, the 2.1-kb BamHI fragment that included this ORF contained a presumptive gapB gene, encoding GAPDH (Gibson and Tabita,1988). Since the deduced amino acid sequence U1 kb

prkB

f bpB

tklB

gapB

SP

"

c

_

"

"c.

"""-c"

rbpL

cfxa

X BPE

Sm

P

xs

P

c)

"-

" "

"t _

c

FIG. 1. Physical and genetic map of the form I1 COz fixation gene cluster in R. sphaeroides and sequencing strategy for the4.1-kb EcoRI fragment betweenprkBand rbpL.Gene designations are given above each open reading frame indicated by boxes. Arrows denote the direction and extentof sequence from either commercial (-+) or custom (k) primers. Restriction sites are: B , BarnHI; X , X h I ; Sm, S m I ; P, PstI; S , SalI; E, EcoRI.

20449

of this ORF shows a high degree of homology to other reported GAPDH sequences and this ORF would encode a 36,000 M, protein whose appearance correlates with increased GAPDH activity (Table I), we conclude that this ORF does indeed code for GAPDH. The sequence of GAPDH is well conserved among diverse species and can be divided into two independent domains. The amino-terminal domain is involved with coenzyme binding whereas the carboxyl-terminal region is concerned with catalysis (Biesecker et al., 1977). The aminoterminus of GAPDH from several sources and other ADP-binding enzymes has been reported to contain the “Bap-foldfingerprint.”

The fingerprint actually describes the positions of 11essential amino acids which allow for nucleotide binding at the @a& fold (Wierenga et al., 1986). We found that positions 4, 5, 7, 9,11, 14,18,21,32,34, and36 of the R. sphaeroides GAPDH were occupied by all 11 essential amino acids (Fig. 6, top). Indeed, this information provides further evidence for the ORF start site for gap& In addition, amino acids which have been previously identified to be important for catalysis such as Cys-152 (Mougin et al., 1988), His-179 (Soukri et al., 1989), Gly-9, Asp-36, Ser-151, Thr-153 (Biesecker et al., 1977; Harris et al., 1976) are all found within the R. sphaeroides GAPDH sequence. Another important region of conservation in

Transketolase and Other E 'nzymes of the Calvin Cycle

20450

* *

* TKL

116

E V T T G P L G Q G I

DHAS

124

E V T T G P L G Q G I G X X G X G

Consensus

* *

*

TKL

456

T H D S I G L G E D G P T H Q P V E

DHAS

484

T H D S I N E G E N G P T H Q P V E

Consensus

G X G X X G FIG.5. Conserved regions in transketolase (TKL) of R.

sphaeroides and dihydroxyacetone synthase (DHAS) of H. polymorpha (Janowicz et al., 1985) that contain potential nucleotide-binding sites. Numbers to the left of each sequence refer to thefirst amino acid residue of each region. Asterisks (*) refer to G residues thought to be important for nucleotide binding (Wierenga et al., 1986).

TKL

FIG.3. Dot plot of deduced amino acid sequences of dihydroxyacetone synthase transketolase ( T K L ) and (DHAS).The amino acid sequences of transketolase and dihydroxyacetone synthase were analyzed by the COMPARE (Wisconsin GCG, Madison, WI) program with a window of 30 and a stringency of 14.

TABLE I Enzyme activity in extracts of E. coli JM107 harboring different plasmid constructs after inductionwith IPTG E. coli JM107 cells containing the indicated plasmids were grown to a visible turbidity using an inoculum from an overnight culture. 1 mM) was added where required and thecells allowed Then IPTG (to to induce for 4-4.5 h. Specific activity" Plasmid"

GPDH Transketolase ~

~

~~

pmol/min/mg protein

None 20.2 0.020 16.5 pJG5 0.019 pJG5.1 0.141 48.0 D J G ~(-IPTG) .~ 27.5 0.021 a Plasmid pJG5 contains the 4-kb EcoRI insert of plasmid pJG106 (Gibson and Tabita,1987). Plasmid pJG5.1 contains the same EcoRI fragment but in the opposite orientation with respect to the loc promoter.

sequence in mustard with other GAPDH sequences, Martin and Cerff (1986) found that thechloroplast enzyme exhibited a higher similarity to the sequences from B. stearothermophilus and Thermus aquaticus than to the cytosolic enzyme. The cytosolic GAPDH demonstrated higher homology to the animal and yeast GAPDH sequences than to thechloroplast enzyme from the same organism. Similar results were found with both the chloroplast and cytosolic GAPDH sequences of tobacco (Shin et al.,1986). This suggested to these authors that chloroplast gap was initially located in thegenome of an endosymbiotic chloroplast ancestor (i.e. photosynthetic prokaryote) and was later transferred intothe nuclear genome of the host during the course of evolution (the endosymbiotic theory). However, the sequence from one important and key group of organisms was missing from these comparisons, the FIG.4. Amino acid sequence alignment of the gene GAPDH sequence from a photosynthetic bacterium. Thus, it product (TKLB) of R. sphaeroides with dihydroxyacetone is interestingthat comparisons of the deduced GAPDH amino synthase (DHAS) from H. polymorpha. Identical amino acids acid sequence from the photosynthetic bacterium R. sphaeroides to sequences from eukaryotes shows 45-48% identity are shaded. while there is 51-60% identity with other known prokaryotic GAPDH is the so-called "S-loop" region (amino acids 178- and chloroplast GAPDH sequences. Although GAPDH has 201 in the Bacillus stearothermophilus enzyme). This internal historically been employed to ascertain evolutionary relatedsequence is important for interactions between the coenzyme ness due to strong conservation of this protein at both primary and neighboring subunits (Biesecker et al., 1977). Clear ho- and tertiary structure levels (Martin and Cerff, 1986), one mology was found between amino acid residues 180 and 203 must be cognizant that accurate evolutionary trees cannotbe of the R. sphueroides deduced GAPDH sequence and the S- drawn from the sequence of one protein. It is readily apparent loop sequence of other bacterial enzymes (Fig. 6, bottom). that GAPDH's from R. sphueroides, chloroplasts, and the In comparing both the cytosolic and chloroplast GAPDH other bacteria share more homology than the enzyme from

Transketolase and OtherEnzymes of the Calvin Cycle

20451

glycolytic gene cluster situated adjacent to genes encoding phosphoglycerate kinase and GAPDH (Alefounder et al., 1989). It is interesting that the putative aldolase in the R. sphaeroides form I1 CO, fixation gene cluster is also adjacent to a gap coding sequence. Expression of t k l B and gapB Gene Productsin E. coli-To furthercharacterize the gene products of tklB and gapB, subclones containing these genes in pUC expression vectors were transformed into E. coli JM107. Plasmids pJG5 and pJG5.1 (Gibson andTabita, 1988) bothcontain the same approximate 4-kbEcoRI fragment of R. sphaeroides DNA but in opposite orientations with respect to the vector promoter. R. rphaeroides Theseconstructs were used to test for the production of transketolase activityin E. coli; extracts were made from cells 2 . mbllls containing each of the plasmids after incubation with IPTG r . aquaticus to induce transcription from the lac promoter. Optimization FIG.6. Comparison of key sequences of GAPDH from di- of the transketolase assay conditions with extracts from R. verse sources. Top, homology between the amino acid terminus of sphaeroides indicated that theR. sphaeroides enzyme did not R. sphmroides GAPDH to other GAPDH amino acid sequence "fin- require exogenous thiamine pyrophosphate or Mg"', unlike a gerprints" with nucleotide-binding properties (Wierenga et al., 1986). commercial preparation of yeast transketolase. Assays of E. Amino acids that are in agreement with the fingerprint are shown coli extracts showed that the R. sphaeroides tklB gene was (*). Abbreviations: x, K, R, H, S, T,Q,N , y , A, I, L,V, M, C; z , D, E. expressed in E. coli from the lac promoter in an orientationNumbers to the left of each sequence refer to the first amino acid dependent fashion (Table I). Activity was obtained only in residue of the region from each source of enzyme. Zymomonus mobilis cells containing plasmid pJG5.1 and was dependent on IPTG (Conway et al., 1987); B. stearothermophilus (Branlant et al., 1989); induction. Furtherstudies indicated that the activity was T. aquaticus (Hocking and Harris, 1980); Saccharomyces cereuisiae xylulose 5-phosphatedependent, with little or no activity (Holland and Holland, 1979); human muscle (Nowak et al., 1981); mustard (Martin and Cerff, 1986). Bottom, homology of bacterial S- obtained with ribulose 5-phosphate as a potential glycoaldeloop regions. Identical amino acid residues are shaded in the top and hyde donor. In addition, active E. coli extracts contained a protein of about M , 68,000, the expected size of the tklB gene. bottom panels. This protein is not found in extracts of cells not containing the tklB gene, in cells containing the gene in the incorrect B 70 orientation, or incells not incubated with IPTG (Fig. 8). The A 70 expression data (Table I, Fig. 8), in conjunction with the sequence homology results (Figs. 3 and 4), strongly suggest B A 140 140 A E that the sequence between p r k B and gapB encodes transketolase. 210 210 ::Q In addition to transketolase, plasmid pJG5.1 also contains .,.,.," ... .. . .." gapB. Work from an earlier study had shown that a 37,000 280 B L M , protein was expressed in E. coli harboring plasmid pJG549 280 A P (Gibson and Tabita, 1988), which contains the presumptive gapB gene, a small portion of tklB (ORF3), andmuch of cfxB. B 350 A 350 Examination of the proteins expressed by plasmids pJG5.1 (Fig. 8, lane 5) indicated that there was a 37,000 M,protein B $KT 3 5 4 A PAY4 3 5 4 produced by these cells; this is about the M, of previously FIG.7. Amino acid sequence alignment of the gene products reported GAPDH from both prokaryotes and eukaryotes. Due of cfxB and cfxA. Identical amino acid residues are shaded. B, cfxB to thehigh endogenous E. coli GAPDH activity, it was difficult gene product; A, c f r A gene product (Gibson et al., 1991). The deduced to measure the activity of the recombinant enzyme; however, cfxB sequence beyond residue 297 is taken from Wagner et al. (1988). we consistently found levels 2-4-fold above the endogenous E. coli activity (TableI); in addition,there is obviously a mammals, chicken, Drosophila, plant cytosol, and yeast, seem- significant amount of recombinant protein synthesized (Fig. 8, lune 5). Whetherthis relatively low (2-4-fold)level of ingly in support of the endosymbiont theory. The ORF corresponding to cfxB begins 81 nucleotides induction reflects unknown assay requirements for the R. downstream from gapB and extends through the EcoRI site sphaeroides GAPDH, the processing of the recombinant en(Fig. 2). The COOH-terminal portion of the cfxB sequence zyme in E. coli, or its inherent instability in this environment, can be deduced from the sequence 5' to rbpL that was deter- remains to be established. It should also be noted that the mined previously (Wagner et al., 1988). The 3'-end of cfxB tklB and gapB genes appeared to be cotranscribed in E. coli extends 165 nucleotides beyond the EcoRI site, terminating since both proteins were expressed only when the cells were 26 nucleotides upstream from rbpL (Fig. 2). The polypeptide induced (Fig. 8, lane 5). These results strongly suggest that encoded by cfxB is 354 amino acids in lengthwith a calculated the two genes may be cotranscribed in the B gene cluster in molecular weight of 38,266. Alignment of the cfxB predicted R. sphaeroides and are compatible with recent findings obamino acid sequence with that of c f d , its homolog from the tained from Tn5 insertional mutagenesis of the B cluster form I COz fixation gene cluster, revealed striking similarity (Gibson et al., 1990). (Fig. 7). The two gene products share 81% identity at the In summary, our results indicate that there are three addiamino acid level. Recently, the cfwi gene product was tenta- tional Calvin cycle structural genes within the cfxB gene tively identified as analdolase because of sequence similarity cluster, beyond what was previously reported (Gibson and to an E. coli class I1 fructose 1,6-bisphosphate aldolase, en- Tabita, 1988). These three genes encode transketolase, glyccoded by the fda gene (Gibson et al., 1991). On the basis of a eraldehyde phosphate dehydrogenase, and aldolase. To our similar gene alignment of the cfxB gene product with the E. knowledge, this study provides the first sequence information coli fda coding sequence, we conclude that cfxB also encodes for prokaryotic transketolase and probably the initial primary aldolase. The E. coli aldolase gene was isolated as part of a structure determination of transketolase from any photosyn,

20452

Transketoluse and Other Enzymes

ofCalvin the

Cycle

between the methylotrophic and COaassimilatory transketolase enzymes. The expression of high levels of the prokaryotic enzyme in E. coli should provide an excellent foundation for detailed structure-function studies of this enzyme and comparisons of conserved residues in the R. sphueroides and H. polymorph enzymes should facilitate site-directed mutagenesis studies. The R. sphaeroides glyceraldehyde phosphate dehydrogenase shows all the requisite key functional residues previously determined from various sources of this enzyme. The homology between bacterial and chloroplast GAPDH sequences, compared to eukaryotic cytosolic enzymes, is consistent with current theories of the evolutionary development of these organisms (Brinkmann et al., 1989). Finally, it should be noted that theorganization of the two distinct chromosomal Confixation gene clusters of R. sphueroides differs only by the presence of the tklBgapB sequences in the B cluster. The physiological consequence of deletion and/or inactivation of the tklBgupB genes in the form I1 or B COz fixation operon is the subject of on-going investigations. Acknowledgments-We are pleased to acknowledge the technical assistance of Robert Sharp and Julie Erickson, and we thank Kathleen Kendrick for her helpful commentsand cooperation. REFERENCES Alefounder, P. R., Baldwin, S. A., Perham, R.N., and Shorts, M. J. (1989) Biochem. J. 257,529-534 Biesecker, G., Harris, J. I., Thierry, J. C., Walkter, J. E., and Wonacott, A. J. (1977)Nature 266,328-333 Branlant, C., Oster, T., and Branlant, G. (1989)Gene (Amst.) 75, 145-155 Brinkman, H., Cerff, R., Salomon, M., and Soll, J. (1989)Plant Mol. Biol. 13, 81-94 Bystrykh, L. V., Sokolov, A. P., and Trotsenko, Y. A. (1981)FEBS Lett. 132, 324-328 Conway, T., Sewell, G. W., and Ingram, L. 0.(1987)J. Bacteriol. 169,56535662 Davis, R. W., Botstein, D., and Roth, J. R. (1980)Aduanced Bacteriol Genetics: A Manual for Genetic Engineering, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY Gibson, J. L., and Tabita, F. R. (1977)J. Biol. Chem. 252,943-949 Gibson, J. L., and Tabita, F. R. (1986)Gene (Amst.) 44,271-278 Gibson, J. L., and Tabita, F. R. (1987)J. Bacteriol. 169,3685-3690 Gibson, J. L., and Tabita, F. R. (1988)J. Bacteriol. 170, 2153-2158 FIG. 8. Analysis of polypeptides produced in E. coli harbor- Gibson, J. L., Chen, J.-H., Tower, P. A., and Tabita,F. R. (1990)Blochemistry 29,8085-8093 ing plasmids containing t k l B andgapB. Sodium dodecyl sulfateGibson, J. L., Falcene, D. L., and Tabita, F. R. (1991)J. Biol. Chem. 266, to analyze plasmid-dipolyacrylamide gel electrophoresis was used 14646-14653 rected protein synthesis in E. coli. Lanes I and 6, commercial (BioHallenbeck P. L., and Kaplan, S. (1987)J. Bacteriol. 169,3669-3678 to bottom: phosphorylase B Harris, J. I.: and Waters,M. (1976)The Enzymes, Vol. XIII, pp. 1-49,Academic Rad) molecular weight markers, from top (M, = 97,400), bovine serum albumin (MI= 66,200), ovalalbumin (M, Press, New York ,J D and Harris, J. I. (1980)Eur. J. Biochem. 108,567-579 = 45,000), carbonic anhydrase (Mr= 31,000), soybean trypsin inhib- Hockin Hollancf J: P.:'and Holland, M. J. (1979)J. Bid. Chem. 254,9839-9845 itor (M, = 21,500), and lysozyme (M, = 14,400); lane 2, lysate of E. Janowicz, 2. A., Eckart, M. R., Drewke, C., Roggenkamp, R. 0..Hollenberg, C. P., Maat, J., Ledeboer, A. M., Visser, C., and Verrips, C. T. (1985)Nucleic coli JM107; lane 3, lysate of E. coli JM107(pJG5); lune 4, lysate of E. Acids Res. 13,3043-3061 coli JM107(pJG5.1) without IPTG induction; lune 5, lysate of E. coli Jordan, D. B., and Ogren, W. L. (1981)Nature 291,513-515 JM107(pJG5.1) induced with IPTG. In lune 5, the position of the Jouanneau, Y., and Tabita, F. R. (1986)J. Bacteriol. 165,620-624 the upper Kato, N., Higuchi, T., Sakazawa, C., Nishizawa, T., Tany, Y., and Yamada, H. presumptive recombinant transketolase is indicated by arrow and the presumptive recombinant GAPDH is indicated by the (1982)Biochim. Biophys. Acta 715, 143-150 Markwell, M. A,, Haas, S. M., Bieber, L. L., and Tolbert, N. E. (1978) AM^. lower arrow. Biochem. 87,206-210 Martin, W., and Cerff, R. (1986)Eur. J. Biochem. 159,323-331 A., Corbier, C., Soukri, A,, Wonacott, A., Branlant, C., and Branlant, thetic or autotrophic organism. In methylotrophic yeasts,the Mougin, G. (1988)Protein Eng. 2, 45-48 oxidation and assimilation of methanol provides energy and Muller, E. D., Chory, J., and Kaplan, S. (1985)J. Bacteriol. 161,469-472 the necessary carbon for biosynthesis. A key enzyme in this Nargang, F., McIntosh, L., and Somerville, C. (1984)Mol. Gen. Genet. 193, 220-224 one-carbon metabolic pathway is the assimilatory enzyme Nowak, K., Wolny, M., and Banas, T. (1981)FEES Lett. 134,143-146 dihydroxyacetone synthase, which is encoded by the d m gene Quivey, R. G., Jr., and Tabita,F. R. (1984)Gene (Amst.) 31,91-101 A. M., and Tabita, F. R. (1989)J. Gen. Microbiol. 135,1699-1713 (Janowicz et al., 1985). This enzyme catalyzes the transferof Rainey, Shih, M.-C., Lazar, G., and Goodman, H. M. (1986)Cell 47,73-80 a glycoaldehyde group from xylulose 5-phosphate to formal- Soukri. A.. Mouein. A.. Corbier. C.. Wonacott.. A,.. Branlant.. C.,. and Branlant, G. (i989)BiocYhemistry 28,258612592 dehyde, generated from the oxidation of methanol (Bystrykh Tabita, F. R. (1987)in The Cyanobacteria (Fay, P., and Van Baalen, C., eds) et al., 1981; Kat0 et al., 1982), to form one molecule each of DD. 95-117.Elsevier Science Publishers B.V.. Amsterdam R.'(1988) Microbiol. Reu. 52, 155-189 Tibita, F. dihydroxyacetone and glyceraldehyde 3-phosphate. Indeed Tahita., ~. F. R.. and ~.. , McFadden. B. A. (1974)J. Biol. Chem. 249.3459-3564 the only difference from the more traditional transketolase of Vira,J., and Messing, J. ( 1 9 8 2 ) C e k ( A k s t . ) 19,259-268 ' the oxidative and reductive pentose phosphate pathways is Wagner, S.J., Stevens, S. E., Jr., Nixon, B. T., Lambert, D. H., Quivey, R. G., Jr., and Tabita, F. R. (1988)FEMS Microbiol. Lett. 55,217-222 the use of formaldehyde as a unique glycoaldehyde acceptor. Wierenga, R. K., Terpstra, P., and Hol, W. G. J. (1986)J. Mol. Btol. 187,101107 Thus, it is not surprising that there is significant homology ~

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