Bradley J. Baker, Joe E. Dotzlaf, and Wu-Kuang YehS. From the Lilly Research ... ium (8, 9); (iii) isopenicillin N epimerase from S. clavuligerus. (10) and S.
Vol. 266, No. 8, Issue of March 15, pp. 5087-5093,1991 Printed in U.S.A.
THEJOURNALOF BIOLOGICAL CHEMISTRY
0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.
DeacetoxycephalosporinC Hydroxylase of Streptomyces clavuligerus PURIFICATION, CHARACTERIZATION, BIFUNCTIONALITY, AND EVOLUTIONARY IMPLICATION* (Received for publication, August 27, 1990)
Bradley J. Baker, JoeE. Dotzlaf, and Wu-Kuang YehS From the Lilly Research Laboratories,A Diuision of Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana46285
Deacetoxycephalosporin C hydroxylasefrom cell- Aspergillus nidulans (4), (ii) isopenicillin N synthetase from free extractsof Streptomyces clavuligerus was stabi- Streptomyces clavuligerus ( 5 ) , Streptomyceslipmanii (6), lized partially and purified to near homogeneity by Streptomyces lactamdurans(7), and Cephalosporium acremonthree anion-exchange chromatographies, ammonium ium (8,9); (iii) isopenicillin N epimerase from S. clavuligerus sulfate fractionation, and two gel filtrations. The hy- (10) and S. luctamdurans (11); (iv) deacetoxycephalosporin droxylase wasa monomer with aM, of 35,000-38,000. synthase from S. lactamdurans (12) and S. cluvuligerus (13, a-Ketoglutarate, ferrous iron, and molecular oxygen 14); and (v) deacetoxycephalosporin C synthetase/hydroxylwere required for theenzyme activity. The hydroxyl- ase from C. acremonium (15, 16). Using “reverse genetics,” ase was optimally active between pH 7.0 and 7.4 in a 3-(N-morpholino)propanesulfonicacid buffer and at structural genes encoding for five isopenicillin N synthetases 29 “C. It was stimulatedby a reducing agent, particu- (S. cluvuligerus, S. lipmanii, C. acremonium, A. nidulans, and larly dithiothreitol or reduced glutathione, and ATP. Penicillium chrysogenum;Refs. 7, and 17-19), for isopenicillin The requirement for ferrous ion was specific, and at N epimerase (20), and for deacetoxycephalosporin C synthase (21) and deacetyoxycephalosporin C synthetase/hydroxylase least one sulfhydryl group was apparently essential for values of the (22) have been cloned and expressed in Escherichia coli. the enzymatic hydroxylation. The K,,, hydroxylase for deacetoxycephalosporin C and a-ke- Complete nucleotide sequences for the eight cloned genes of toglutarate were 59 and 10 BM, respectively, and the p-lactam biosynthesis have now been determined. K,, for ferrousion was 20 BM. In addition to itsknown In addition to their value in understanding the regulation hydroxylation of deacetoxycephalosporin C to deace- and evolution of the two p-lactam biosynthetic pathways, the tylcephalosporin C, the hydroxylase catalyzed effec- cloned genes have practical applications to problems in the tively an analogous hydroxylation of 3-exomethyl- synthesis of cephalosporin antibiotics (3). Introductionof an enecephalosporin C to deacetoxycephalosporin C. Sur- extra copy of deacetoxycephalosporin C synthetaselhydroxprisingly, the hydroxylase also mediated slightly a novel ring-expansion of penicillin N to deacetoxyce- ylase gene to an industrial production of C. acremonium phalosporin C. The substratespecificity of the hydrox- resulted in a 15% increase in the synthesis of cephalosporin that C (23). Deacetoxycephalosporin C can be enzymatically deylase is overlapping with but distinguishable from of deacetoxycephalosporin C synthase, the enzyme acylated to form 7-aminodeacetoxycephalosporanic acid (24), which normally mediates the ring-expansion reaction an important intermediate in the manufacturing of oral ceph(Dotzlaf, J. E., and Yeh, W. K. (1989)J. Biol. Chem. alosporin antibiotics. Cloning of both isopenicillin N epimerase and deacetoxycephalosporin C synthase genes of S . cla264, 10219-10227). Furthermore,thehydroxylase exhibited an extensivesequence similarity to the syn- vuligerus to P. chrysogenummay lead to anindustrially useful thase. Thus, the two enzymes catalyzing the consecu- recombinant strain for deacetoxycephalosporin C biosyntive reactions forcephamycin C biosynthesis in S. cla- thesis (Fig. 1;Ref. 3). This is because P.chrysogenum is vuligerus represent apparent products from a diver- capable of producing more of the precursor isopenicillin N gent evolution. than C. acremonium. Expression of the S. clavuligerus synthase gene in P. chrysogenum has been achieved although a relatively low-level of the enzyme activity was obtained (25). Since deacetoxycephalosporin C synthetase/hydroxylase of Cephalosporin biosynthesis has received an increasing sci- C. acremonium is a bifunctional enzyme (15, 22), an alternaentific attention in the last few years. This is due to a tive route for biosynthesis of deacetoxycephalosporin C using combination of the important therapeutic value of cephalo- C. acremonium would require specific inactivation of the sporin antibiotics and the application of recombinant DNA hydroxylase activity to allow deacetoxycephalosporin C actechniques to filamentous fungi and streptomyces (1-3). Five cumulation in vivo (Fig. 1).Separability of the deacetoxycefunctionally different enzymes that are common for cephalophalosporin C synthase and hydroxylase activities of S. clasporin C and cephamycin C biosyntheses (Fig. 1) have been purified to near homogeneity from bacteria and/or fungal vuligerus (26) provides an invaluable opportunity to elucidate synthetase from the functional domains of the two enzymes. This could greatly sources: (i) L-a-aminoadipyl-cysteinyl-valine facilitate the identification and specific mutagenic inactiva* The costs of publication of this article were defrayed in part by tion of the hydroxylase activity of the bifunctional C. acrethe payment of page charges. This article must therefore be hereby monium enzyme. We describe here the purification to near marked “aduertisement” in accordance with 18 U.S.C. Section 1734 homogeneity of deacetoxycephalosporin C hydroxylase from solely to indicate this fact. The paper is dedicated to the memory of Dr. Dave W. Dennen, an S. cluvuligerus and the characterization of its essential properties, including a novel substrate specificity. We also discuss inspiring leader of Biotechnology. $To whom correspondence should be addressed. Tel.: 317-276- a surprisingfunctional/evolutionary relationship between the 7796. hydroxylase and the synthase.
5087
DeacetoxycephalosporinC Hydroxylase of S. clavuligerus
5088 L-a.Amlnoadiplc Acid
+
I
+
L-Cysteine
L.Vallne
TABLE111 Substrate specificity of DAOC hydroxylase Purified enzvme (4.5 milliunits) was used in each assav.
Synthry
@-Lactam
I 1
Relative activity %
For hydroxylation DAOC EMCC ISO-DAOC" Z-CH,-DAOC Sulfone DAC Cephalosporin C For ring-expansion: Penicillin N
Cyclase
Epimerase
100 37 0 0 0 0 3
With L-form side-chain (D-form side chain in Fig. 1).
I
I
COzH
L-""""""""""""""""""""""""""!
8 I
A
Acetyltransferase
/\
Carbarnoyltransferase 7.Hydroxylase 7.O.Mathyltranslerase
COzH Cephalosporin C
Cephamycin C
FIG. 1. Cephamycin C/Cephalosporin C biosynthesis. EXPERIMENTAL PROCEDURES AND RESULTS'
Substrate Specificity-The 3'-hydroxylation of EMCC,' a P-lactam analogue of the natural substrate DAOC was examined under conditions supporting optimal activity of the highly purified hydroxylase (Porous Q eluate, Table I). Aside from theexpected DAOC +DAC conversion, the hydroxylase also catalyzed the conversion of EMCC to DAC (Table 111). The hydroxylation activity with EMCC as substrate was about one-third of that withDAOC. No hydroxylation activity with iso-DAOC or 2-CH3-DAOC sulfone was detectable. In addia slight tion to the 3'-hydroxylation of the two cephalosporins, ring-expansion activity was observed in the penicillin N + DAOC + DAC conversions (Table 111). Enzyme Inhibition-When DAOC was used as the substrate at 0.1 mM, the hydroxylase was inhibited a t 21, 62, and 100% by 0.1, 1, and 10 mM penicillin N, respectively. With EMCC Portions of this paper(including "Experimental Procedures," part of "Results," Figs. 2-6, and TablesI and 11) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that available is from WaverlyPress. The abbreviations usedare: EMCC, 3-exomethylenecephalosporin C: DAOC, deacetoxycephalosporin C; DAC, deacetylcephalosporin C; a-KG, a-ketoglutarate;DTT,dithiothreitol;p-HMB, p-hydroxymercuribenzoate; NEM, N-ethylmaleimide; DTNB, 5,5'dithiobis-2-nitrobenzoic acid; MOPS, 3-(N-morpholino)propanesulfonic acid; HPLC, high performance liquid chromatography; FPLC, fast protein liquid chromatography; and SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
l/(DAOC), mM-1
FIG. 7. Inhibition of DAOC hydroxylase by penicillin N. The purified hydroxylase (Porous Q eluate, 10 pg of protein, Table I) was analyzed a t 0 (O),0.5 (O), and 1 m M (X) penicillin N with DAOC as the variable substrate. The mode of inhibition was evaluated by the COMPO program of Cleland (31).
as the substrate at 0.1 mM, the enzyme was inhibited at 15 and loo%, respectively, by 1 and 10 mM penicillin N. The inhibition of the hydroxylase by penicillin N was competitive with DAOC as the variable substrate (Fig. 7). The Kifor penicillin N, as determined from the slope replot, was 0.12 mM. Effects of Metal Ions and Reducing Agents-Fe2+ was required for expression of maximal activityof the highly purified DAOC hydroxylase. It could not be replaced by M$+, Mn2+, Co2+,Ca2+, Cu2+, Ni2+, Zn2+,Na+, or K+. Very little hydroxylase activity ( Co2+> Niz+, MnZ+. Effects of Metal Chelators and Sulfhydryl Reagents-DAOC hydroxylase was highly sensitive to inhibition by o-phenanthroline and moderately inhibitedby EDTA (Table IV). The hydroxylase was also very susceptible t o inhibition by sulfhydryl reagents according to the order: p-hydroxymercuribenzoate > 5,5'-dithiobis-2-nitrobenzoicacid, N-ethylmaleimide > iodoaceticacid. The enzyme inhibition by DTNB was completely reversible by further addition of DTT (data not shown). Immunological Cross-reactivity-A positive immunological cross-reaction was detectedamongthe fungal synthetase/
DeacetoxycephalosporinC Hydroxylase of S. clavuligerus TABLEIV Effects of metal chelators and sulfhydryl reugentson DAOC hydroxylnse
5089
TABLEV DAOC synthetuse/hydroxyluse, DAOC synthnse, and DAOC hydroxylase: activity comparisonwith three &lactam substrates The relative activitiesfor the synthase and the synthetase/hydroxylase are from unpublished data (J. E. Dotzlaf and W.-K. Yeh) and
Enzymic reactions for the hydroxylasewereconductedwith a partially purified enzyme (0.23 milliunits/assay) in the presence of 0.08 mM FeSO, and 1 mM DTT. The enzyme was incubated with an those for the hydroxylase are fromthis report. inhibitor for 1 min prior to reaction initiation with DAOC. p-HMB, Relative activity p-hydroxymercuribenzoate; DTNB, 5,5'-dithiobis-2-nitrobenzoic 3'-Hydroxylation Ring-expansion, Enzyme acid. Penicillin N
Concentration activity Addition Relative mM
None o-Phenanthroline EDTA p-HMB
0.05 0.5 0.05 0.5 0.01
+ DAOC
DAOC
~
DAC EMCC
"f
DAC
%
100 12 0 51 0
Synthetase/hydroxylase Synthase Hvdroxvlase
57
100
83
52 3
0.8 100
23
-
0.6 37
DAOC and 3'ality (ie. ring-expansion of penicillin N hydroxylation of DAOC + DAC) was proven conclusively 1 0 from copurification of the two activities (15) and subsequent 0.1 64 N-ethylmaleimide expression of both activities from the cloned gene in E. coli 1 0 (22). Also, the 3'-hydroxylation of EMCC wasreadily detect1 19 Iodoacetic acid 10 0 ablewiththefungalexpandase/hydroxylase (32) and the DAOC synthase recombinant enzyme expressed in E. recently purified to near homogeneity from S. lactamdurw Synthase R..ldu.. 1.28 ~ T ~ T ~ S ~ E [ f l Q Q [ I l ' H ~ was D q D ans (12) and S. clauuligerus (13, 14). Both synthases were Hydroxylase A D T P V P I F N L A A L R E G A D Q E K F R E C V T G shown clearly to catalyze the ring-expansion of penicillin N Synthetase, M T S K V P V F R L D D L K S G K V L T R L A E A V T T --f DAOC. DAOC synthase from S. clauuligerus did not cataHydroxylase lyze the hydroxylation of DAOC + DAC, it did mediate the (6)Rnlduw 01-98 hydroxylation of EMCC + DAC at a low level (14). Our Synthase IT G S Y ~ S D Y S MI recentanalysisusing ahighly activerecombinant DAOC synthase, however, allowed detection of a slight enzymatic Hydroxylase hydroxylation of DAOC + DAC (Table V). Thus, the prokarFIG. 8. Sequence comparisons among DAOC hydroxylase, yotic DAOC synthase is alsoendowed with a limited hydroxDAOC synthase, and DAOC synthetase/hydroxylase. A , 28- ylation capability. On the other hand, DAOC hydroxylase was residue amino-terminal sequences; identical residues from at least effective in mediating the hydroxylation of not only DAOC two of the three sequencesare enclosed inboxes. B, %residueinternal DAC but also EMCC + DAC. Unexpectedly, the hydroxsequences; all identical residues from at least two of the three seylase also catalyzed slightly the ring-expansion of penicillin quences are enclosed in a single solid box. N to DAOC that is furtherhydroxylated to DAC by the same enzyme. Whether the ring-expansion activity represents an hydroxylase, the bacterial synthase, and the bacterial hydroxof the hydroxylase or is due to a minor intrinsic property ylase (data not shown). contamination from the synthase cannow be answered. The S. cluuuligerus hydroxylase gene hasbeen cloned andexDISCUSSION pressed in E. coli as an insoluble form in granules (36). In of DAOC + DAC, the Enzyme Purificationand Function Elucidation-This is the addition to the expected hydroxylation first report describing the purification of DAOC hydroxylase recombinant hydroxylase also catalyzed the ring-expansion DAOC at an extent identical to that of the from any cephamycin C-producing Streptomyces species. Crit- of penicillin N ical aspects in the hydroxylase purification procedure were native enzyme (TablesI and 111). the timely harvest of S. cluuuligerus cells with maximal enThus, the bacterial DAOC synthase andDAOC hydroxylase zyme activity, controlled cell disruption by sonic treatment are bifunctional enzymes just like the fungal DAOC synthe(lo), a partial hydroxylase stabilization in the cell-free ex- tase/hydroxylase.Basedon relativeenzyme activities observed under conditionsoptimized for the ring-expansion and tracts, and an optimized chromatographic enzyme purification. The hydroxylase from Porous Q eluate was 92% pure hydroxylation reactions, respectively (Table V), the hydroxand highly active with a VmaX of 0.49 units/mg (Table I, Fig. ylase functions much more like the synthetase/hydroxylase 3). The minor protein, that accounted for the remaining 8% than itdoes the synthase. of the total protein was consideredto bea degradation product Physical, Catalytic, and Kinetic Properties-DAOC hydroxof the native hydroxylase protein, based on a high sequence ylase of S. clauuligerus shares an extensive similarityDAOC to similarity of its 9-residue amino-terminalsequencetoan synthetase/hydroxylase ofC. acremonium and DAOC syninternal 9-residue sequence from both the bacterial DAOC thase of S. clauuligerus in its physical, catalytic, and kinetic synthase (21) and the fungal DAOC synthetase/hydroxylase properties (13-16,21-22). The M , of the hydroxylase (35,000(22), as shown in Fig. 8. A broad single protein band was 38,000) is very similar to that of the synthetase/hydroxylase observed after native-polyacrylamide gel electrophoresis (Fig. (36,500-41,000) and to that of the synthase (34,600-36,000). 3B). This enzyme purification provides highly pure and very Highly purified DAOC synthase from S. lactamdurans has a active DAOC hydroxylase t o allow evaluation of its properties lower M,, 27,000 (12). The three different enzymes share an as well as structure-function for comparison to DAOC syn- identical requirement for a-KG, Fe2+, and02.Their requirethase and DAOC synthetase/hydroxylase. ment for Fez+is specific, since no other metal ion (except Fe3+ DAOC synthetase/hydroxylase of C. acremonium was purified previously to near homogeneity (15, 16). Its bifunctionJ. E. Dotzlaf and W.-K. Yeh, unpublished data. DTNB
0.1 0.1
0
64
-
5090
Deacetoxycephalosporin C Hydroxylase of S. cluvuligerus
under reducing conditions) can substitute for Fez+ in expres-droxylations of both DAOC --$ DAC and EMCC + DAC, sion of the ring-expansion or hydroxylationactivity. All three whereas DAOC synthase mediatesmainly the ring-expansion enzymes can be stimulatedgreatly by DTT, but only the of penicillin N --$ DAOC and slightly the hydroxylation of synthetase/hydroxylase and the hydroxylase can be stimuDAOC + DAC and EMCC + DAC (Table V). Thus, funclated by ATP. The stimulationby the reducing agent and the tionally, the threeenzymes can be differentiated by the relasusceptibility to inhibition by a few sulfhydryl reagents sug- tive efficiencies with which they catalyze the ring-expansion gest that at least one sulfhydryl group from each of the three and 3‘-hydroxylation of the three P-lactam substrates. enzymes is essential for catalytic ring-expansion and for hyA Common Active Site for Ring-Expansion and Hydroxyldroxylation. The number and location of the putatively im- ation?-The question of a common active site or two indeportant sulfhydryl residues of the threeenzymes remain to be pendent active sites for thering-expansion and 3”hydroxylelucidated, possibly by chemical modification coupled with ation by the synthetase/hydroxylase, the synthase, and the peptide sequence determination and/or by cysteine-specific hydroxylase is of considerable scientific interest. The ability mutagenesis and activity analysis. The K, values of the three to efficiently synthesize DAOC in C. acremonium by siteenzymes are similar for their respective @-lactam substrates directed inactivation of the hydroxylase activity in the bi(20-25 p ~ and ) the common a-KG (10-22 p ~ ) ,and the K, functional enzyme depends on the answer to this question. valuesfor the common Fez+ (4-20 p M ) are not far apart. The competitive inhibition by EMCC (14) in the synthaseHowever, the hydroxylase can be differentiated readily from catalyzed conversion of penicillin N ”-* DAOC providesa the synthetase/hydroxylase and the synthase in its activities kinetic indication of a common active site for both theenzyfor the 3’-hydroxylation of DAOC + DAC and EMCC + matic ring-expansion and hydroxylation. Similarly, an indeDAC as well as for the ring-expansion of penicillin N + pendent kinetic indicationof a single functional sitemay also DAOC (Table V). be surmised from the competitive inhibition by penicillin N Bifunctionality, A Novel Enzymatic Ring-expansion-The in the hydroxylase-catalyzed conversion of DAOC + DAC. enzymatic conversions of penicillin N ”-* DAOC + DAC and Even though penicillin N was not a strong inhibitor for the of penicillin N + DAOC have been well documented in the hydroxylation reaction, such a competitive inhibition mode last several years with purified DAOC synthetase/hydroxylase was still observed (Fig. 7). The kinetic indication for a com(15,16) andDAOC synthase (13,14),respectively. In contrast, mon active siteof the enzyme, albeit new and interesting, can our observation that DAOC hydroxylase catalyzed the ring- become conclusive only with further structuralelucidation of expansion of penicillin N + DAOC is new (Tables 111and V). the functional site. Since,inadditionto DAOC, EMCCcan serve as a good Apparent Divergent Evolution-Based on the bifunctionalhydroxylation substrate for the hydroxylase (Table 111),we ity of the fungal DAOC synthetase/hydroxylase (15, 22) and speculatethatthe hydroxylasemayalso mediate aringthe separability of DAOC synthase and DAOC hydroxylase expansion of penicillin N + EMCC --$ DAC. Using a n exces- from cell-free extracts of S. cluvuligerus (26), the gene for the sive amount of the highly active enzyme under theoptimized bifunctional fungal enzyme was proposed to arise by a horiconditions for an extended reaction time, EMCC was not zontal fusion-truncation mechanism from two separate genes detected as an intermediate in hydroxylase-catalyzed the con- for procaryotic DAOC synthase andDAOC hydroxylase (22). version of penicillin N -+ DAC. Thus, the capability of the This evolutionary proposal for DAOC synthase/hydroxylase hydroxylase t o catalyze the penicillin N +EMCC conversion could be substantiated by separate ring-expansion and hyremains an open question. As an indication of the catalytic droxylation sites for the bifunctional enzyme as well as by diversity, thepotential hydroxylase-catalyzed ring-expan- two structurally and functionally different DAOC synthase sions and hydroxylations areshown in Scheme 1. In compar- and DAOC hydroxylase enzymes for the ring-expansion and ison, DAOC synthetase/hydroxylase catalyzes effectively the hydroxylation. The first clue in opposition to the hypothesis ring-expansion of penicillin N + DAOC as well as the hy- comes from the similar molecular weights of the three enzymes, i.e. thesynthetase/hydroxylase is not significantly larger than the synthase or the hydroxylase. Currently, there is no direct structuralevidence for or against the separability of the two functional sites in the synthetase/hydroxylase. Pen N However,regardless of theextentincatalytic capability, either DAOC synthase or DAOC hydroxylase has both ringexpansion and 3‘-hydroxylation functions which they share EMCC with DAOC synthetase/hydroxylase (TableV). A kinetic evalSCHEME1. Proposed DAOC hydroxylase-catalyzed ring-ex- uation has indicateda common active sitefor the ring-expanpansion reactions. sion and hydroxylation for the synthase (14) as well as for the hydroxylase (Fig. 7). The detectableimmunological crossTABLE VI reactions among the synthetase/hydroxylase, the synthase, Amino acid composition and sequence comparisons among the and the hydroxylase (data not shown) suggest a structural hvdroxvlase. svnthase and svnthetaselhvdronvlase similarity among the three enzymes. Using the compositional analysis as designated by Marchalonis and Weltman (33), an Enzyme pair SAQ value of 20 as calculated for the hydroxylase and syn% thase (i.e. any SAQvalueless than 50) would indicate a Hydroxy1ase:synthase 20 59 structural similarityof the two enzymesthat could be revealed Hydroxylase:synthetase/hy36 54 SAQ by sequencecomparison. In addition, the smaller an droxylase value can imply a stronger sequence similarity. Both turned Synthase:synthetase/hydroxyl29 57 out to be the case. A significant similarity between the two ase enzymes exists in the %residue amino-terminal sequences, a Calculated from identical residues of approximately 310-residue the %residue internal sequences (Fig. S), andthealmost sequences by the BESTFIT program from the Universityof Wisconcomplete nucleotide-derived sequences (Table VI). A composin ( 3 6 ) .
Deacetoxycephalosporm L Hydroxylase of S. clavuligerus sitional similarity with an SAQvalue of 36 and a significant sequence similarity in thetwo corresponding locations as well as in the almost complete nucleotide-derived sequences are also observed between the hydroxylase and the synthetase/ hydroxylase (Table VI; Fig. 8). Similar composition and sequence similarities also exist for the synthase andthe synthetase/hydroxylase. It is of interest to note, from both the composition and sequence data among the threeenzyme pairs (Table VI), the hydroxylase resembles the synthase the most. The functional and structural comparisons, as described above, strongly suggest that DAOC hydroxylase sharesa divergent evolution with both DAOC synthase and DAOC synthetase/hydroxylase; i.e. the three different enzymes represent evolutionary products from a common ancestral gene. This suggestion can be considered as a revision, based on the recent biochemical data (thisreport; Ref. 14), of the previous gene fusion-truncation proposal (22). The partof the hypothesis for horizontal gene transfer from bacteria to fungi remains valid. Since all three enzymes require a-KG, Fe2+, and 02, it is of scientific interest to analyze whether they are members of an evolutionary family for a-KG-dependent nonheme iron oxygenases for procaryotic/eucaryotic primary metabolism. Also, theapparent divergent evolution between DAOC synthase and DAOC hydroxylase, the enzymes catalyzing two consecutive reactions of cephamycin C biosynthesis in S. clauuligerus (Fig. l),is a novel observation for a specialized/secondary metabolism. The divergent evolution for enzymes mediating two consecutive reactions has been reported for primary metabolisms, e.g. between muconolactone isomerase and @-ketoadipateenol-lactone hydrolase from bacteria (34). The divergent evolutionary relationship for the bacterial synthase and hydroxylase provides a strong support to the “retroevolution” hypothesis of Horowitz (35). It remains to be answered what drives natural selection to the fungal bifunctional synthetase/hydroxylase in contrast to the two independent bacterial enzymes. Acknowledgment-We thank George W. Huffman and Robin D. Cooper for synthesizing and providing all highly pure p-lactam compounds used in this work; Robert E. Weeks for providing S. clauuligerus cells;Adam J. Kreuzman for excellent technical assistance; Robert M. Ellis for amino acid analysis; Dave J . Oakley and Richard M. Van Frank for amino-terminal sequence determination and isoelectric point estimation; Larry D. Tabor and J . Richard Sportsman for polyclonal antibody preparation; Steve Kovacevic and James R. Miller for providing the nucleotide-derived hydroxylase sequence prior to its publication; Eugene T. Sen0 for helpful suggestions in preparation of this manuscript; Stephen W. Queener for advice; and Warren MacKellar and Mark A. Foglesong of Lilly’s management to continuous support on p-lactam biosynthesis. REFERENCES 1. Ingolia, T. D., and Queener, S. W. (1989) Med. Res. Reu. 9, 245264 2. Queener, S. W. (1990) Antimicrob. Agents Chemother. 34, 943948 3. Yeh, W.-K., and Queener, S. W. (1991) Ann. N. Y. Acad. Sci., in press 4. van Liempt, H.,von Dohren, H., and Kleinkauf, H. (1989) J . Bid. Chern. 264,3680-3684 5. Jensen, S.E., Leskiw, B. K., Vining, L. C., Aharonowitz, Y., Westlake, D. W. S., and Wolfe, S. (1986) Can. J . Microbiol. 3 2 , 953-958
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6. Weigel, B. J., Burgett, S. G., Chen, V. J., Skatrud, P. L., Frolik, T. D. (1988) J. Bacteriol. C.A., Queener. S. W., and Ingoha, 170,’3817-3826 7. Ramos. F. R.. LoDez-Nieto.M. J.. andMartin. J. F. (1985) Antimicrob.AgentsChernother. 2 j , 380-387 ’ 8. Hollander, I. J., Shen, Y.-Q., Heirn, J., Dernain, A. L., and Wolfe, S. (1984) Science 2 2 4 , 610-612 9. Pang,C.-P.,Chakravarti, N., Adlington, R.M., Ting, H.-H., White, R. L., Jayatilake, G. S., Baldwin, J. E., and Abraham, E. P. (1984) Biochem.J. 222,789-795 10. Usui, S., and Yu, C.-A. (1989) Biochim. Biophys. Acta 999, 7885 11. Laiz, L., Liras, P., Castro, J. M., Martin, J. F. (1990) J . Gen. Microbiol. 1 3 6 , 663-671 12. Cortes, J., Martin, J. F., Castro, J. M., Laiz, L., and Liras, P. (1987) J. Gen. Microbiol. 133, 3165-3174 13. Rollins, M. J., Westlake, D.W. S., Wolfe, S., and Jensen, S.E. (1988) Can. J. Microbiol. 34, 1196-1202 14. Dotzlaf, J. E., and Yeh, W.-K. (1989)J . Bid. Chem. 2 6 4 , 1021910227 15. Dotzlaf, J. E., and Yeh, W.-K. (1987) J . Bacteriol. 169, 16111618 16. Baldwin, J. E., Adlington, E. M., Coates, J. B., Crabbe, M. J. C., Crouch, N. P., Keeping, J. W., Knight, G. C., Schofield, C. J., Ting, H. H., Vallejo, C. A,, Thorniley, M., and Abraham, E. P. (1987) Biochem. J. 245,831-841 17. Leskiw, B. K., Aharonowitz, Y., Mevarech, M., Wolfe, S., Vining, L. C.,Westlake, D. W. S., and Jensen, S.E. (1988) Gene (Amst.) 62,187-196 18. Samson, S. M., Belagaje, R., Blankenship, D. T., Chapman, J. L., Perry, D., Skatrud, P. L., Van Frank, R. M., Abraham, E. P., Baldwin, J . E., Queener, S. W., and Ingolia, T. D. (1985)Nature 318,191-194 19. Carr, L. G., Skatrud, P. L., Scheetz 11, M. E., Queener, S. W., and Ingolia, T. D. (1986) Gene (Amst.) 48, 257-266 20. Kovacevic, S., Tobin, M. B., and Miller, J . R. (1990) J. Bacteriol. 172,3952-3958 21. Kovacevic, S., Weigel,B. J., Tobin, M. B., Ingolia, T. D., and Miller, J . R. (1989) J. Bacteriol. 171, 754-760 22. Samson, S. M., Dotzlaf, J. E. Slitz, M. L., Becker, G. W., Van Frank, R. M., Veal, L. E., Yeh, W.-K. Miller, J. R., Queener, S. W., and Ingolia, T. D. (1987) Biotechnology 5, 1207-1214 23. Skatrud, P. L., Tietz, A. J., Ingolia, T. D., Cantwell, C. A., Fisher, D. F., Chapman, J. L., and Queener, S. W. (1989)Biotechnology 7,477-485 24. Komatsu, K., Suguira, K., Matsuda, A., and Yamamoto, K. (1988) Jpn. Kokai Tokkyo Kono J P 63 74,488 25. Cantwell, C. A,, Beckmann, R. J., Dotzlaf, J. E., Fisher, D. F., Skatrud, P. L., Yeh, W.-K., and Queener, S. W. (1990) Curr. Genet. 17,213-221 26. Jensen, S. W., Westlake, D. W. S., and Wolfe, S. (1985) J . Antibiot. 38, 263-265 27. Nagarajan, R., Boeck, L. D., Gorrnan, M., Hamill, R. L., Higgins, C. E., Hoehn, M. M., Stark, W. M., and Whitney, J . G. (1971) J . Am. Chem. SOC.93,2308-2310 28. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 29. Sportsman, J. R., Park, M. M., Cheresh, D. A., Fukuda, M., Elder, J. H., and Fox, R. I. (1985) J. Zmmunol. 135, 158-164 30. Blackshear, P. J. (1984) Methods Enzymol. 104, 237-255 31. Cleland, W. W. (1970) in The Enzymes (Boyer, P. D., ed) Vol. 2, pp. 1-65, Academic Press, New York 32. Baldwin, J. E., Adlington, R. M., Schofield, C. J., Crouch, N. P., and Ting, H.-H. (1987) J . Chem. Soc. Chem. Commun. 15561558 33. Marchalonis, J. J., and Weltman, J. K. (1971) Comp. Biochem. Physiol. 3 8 B , 609-625 34. Yeh, W. K., Davis, G . , Fletcher, P., and Ornston, L. N.(1978) J . Bioi. Chem. 253, 4920-4923 35. Horowitz, N.H. (1965) in Euoluing Genes and Proteins (Bryson, V., and Vogel, H. J., eds) pp. 15-23, Academic Press, New York 36. Kovacevic, S., and Miller, J. R. (1991)J . Bacteriol. 173, 398-400 Continued on next page.
5092
Deacetoxycephalosporin C Hydroxylase of S. cluvuligerus SUPPLEMENTARY MATERiALS TO DeaCstoXycephaiospDrin C Hydroxylase of Slrepfomyces clavui,gerus: Purification,Characterization.Bifundionality and Evolutionary implication Bradley B. Baker. Joe E. Dotzlaf, and Wu-Kuang Yeh EXPERiMENTAL PROCEDURES
Materials. Highly purified penicillin N, DAOC. DAC and EMCC (each was 96% pure or greater by HPLC) were available from Eli Ully and Company, Indianapolis, IN. The foilowing materials were purchased from the mmpanles specified: MOPS, a-KG. Dm, ascorbate. PMSF. 0-phenanthroiine, EDTA, p-HMB. NEM. DTNB, Sigma Chemical Co., St. Louis, M O Ultragel AcA54. IBF Biotechnics, Villeneuve-la-Garenne. France' DEAE-Sepharoseand Mono c) Pharmacia Inc. Piscataway NJ' and Poms Q PeAeptive Blosysiems. Cambridge. MA. All other &hemicais were i f the highesipuriy commerciaily avafiaMe.
CNde Eflracl
Organism. An improved cephamycin C-prcducing strain of Sfrepfomyms davuligerur ATCC27064 was grown in a 15-liter fermenter underconditions described previously (27). Cells containing a maximal activity of D A W hydroxylase (about 15 hours aner inoculation) were hawesled by centrifugation. washed with 15 mM MOPS, pH 7.4. in the p r e s e m of 1.0 M KCiand then with the salt-free buffer, and nored at. 70 "C until required.
303
3.571
194
2,919
Fraction
100
1.57
6.57
32
0.02
11.6
23
0.W
15
19
0.02
~ona o Eluate
8.5
1.467
173
9.4
Unragei AcA54 I1Eluate
2.4
732
305
4.7
0.04
Poms a Eluate
0.45
222
493
1.4
0.03
Amino add Asp+Asn Thr Ser GiurGin Pro
2: $2 Met IC Leu Tyr Phe His LYS
Arg Trp
No. 01 residues per 35,000daiton 25 26a 27a
32 18 31 37 5b 21 9
8 23 11 17 8 6 19 2c
aDeiermined byextrapiation 10 zero lime of hydroiysis. bDeterminedas cyslek acid COBtermined by hydrolysis in the presence 01 thioglycokcadd.
ImmunologicalAnalysis. Poiyclonal antibody against purified recombinant fungal deacetoxycephaiorporinC synthetaselhydroxylasewas prepared by L. D. Tabor and J. R. Sponsman of Lilly Wdlanawlis, iN1. The immunolwicA cross-reactionof the hvdmmlase or DAOC Svnthase1141 to this antibody was analyzed by Westein Blotting as previously de&iib& (29) Other Methods. Procedures for SDS-PAGE and moiecuiar weLght determination,and amino acid analysis are those described previously (14). Native-PAGE wasconduned scmding to Biackshear (30).
RESULTS Enzyme Stability. DAOC hydroxylase from crude extracts of S. davdigeNs , as prepared at 4 -C in 15 mM Tris-HCI. pH 7.5, had a half-life of 12 hours Presence 01 PMSF and ethanol in the CNde enracts. which paniaily prnected DAOC synthase from inactivation (14). ais0 paniaily nabilired DAOC hydroxylase. Whenthe crude extracts were prepared in 15 mM MOPS buffer. pH 7.4, In the presence of 10 mM DTT. 1mM PMSF and 10% ethanol. the half-llfe of the hydroxylase improved about IO-fold. Enzyme Purity. DAOC synthase of S. dawligerus was purified 180-foldby a six-step procedure as Summarizedin TaMe I. The hydroxylaseactivity and protein peaks from Porous (1FPLC were m m i y ovedapped (Fig. 2E). The hydroxylaseand residuai Synthase aclivities were inseparable (TaMe 1). When analyzed by SDS-PAGE. the Porous SIM eluate (Table i, step 6 ) migrated as a major protein and a minor protein (Fa.3A).From a laser densitometric scan of the gel, the major protein was 92% pure. An aminoterminal sequence analysis 01 the remaining 8% minor protein (as shown below)suggens it, based on a high internal sequence similarity to the bacterial D A W synthase (21) and to the fungal DAOC SynthelasBhydroxyiase(22). as a degradation product 01 the major protein Only a broad single band was observed from a protein analysis by native-PAGE(Fig.38). Physical Propems The mOlBCUiar weight of the native DAOC hydroxylase. as estimated by gel filtration with Unragel AcAM. was 35.000 (Fig. 4A). The subunit size 01 the enzyme, as determined by SDS-PAGE. was 38,WO (Fig. 48). From the nucleotide sequence of 1% stwcturai gene (J. R. Miller. personal communication), the hydroxylaseshowed a minimal moimlar weight of 34.584. The ikoelactrk point of the enzyme w a s 4.8M.2. Thus. the hydroxylase was an addic monomer during as purification. Amino Acid Composition and Amino-Terminal Sequences. The amino acid oompition Of the D A W hydroxylase is shown in Table Ii.The 28-residue amino-temlnal sequence 01 the native hydroxylase protein and the 9-residue amino-terminalsequence of the degraded hydroxylase protein are shown below.
E
Reaction Stoichiometry. The molar ratio for DAG formationlDAOC disappearance during a 30-min of the hydroxylase-catalyzed reaction was ina range of 0.951.05 (Ftg. 6). The mnvenion of D A W to DAC was complete under the reaction conditions.
55.75% (NHI)ISOI
2.69
Table II. Amino acid mmposition of DAOC hydroxylase from S. daVuf&eNS
After the Sonication.t mM PMSF was added.Ths Supernatant fraction. which was obtained by centnfugation at 47.000 x g lor 30 mm. was used as the crude extract (Table I). The CNdeenracl was applied to a DEAE-Sepharose column (2.6~45cm) previously equliibraied With buffer A. The column was washed witht w o bed volumes of buffer A and boundproteins were eluted Wlth a linear gradient 01 Trisacetate (0-0.9 M) in buffer A. DAOC hydroxylasewas eluted as rwa major activity peaks and well separated from DAOC synthase (Fig, 2A).In addition. a minorsynthase activity coeluled with the larger major hydroxylase activity and, conversely, a minor hydroxylase aclivity coeluted with the main synthase activity (Fig. 2A). The peak fractions from the larger major peak mntaining 32% 01 the loaded hydroxylase activity were pooled (Table 1. step l ) , mncentrated and fractionatedby ammonium sulfale (Table 1. step 2). and one mi 01 5575% fraction loaded onto an Ultragel AcAM column (1xtOO cm) previously equilibratedwith buffer A. Prolein elution was made with buffer A. The hydroxylasewas eluted as a single activity peak (Fig. 28) and the peak fractions mntaining 64% 01 the loaded hydroxylaseactivity were pooled (Table I, step 3) and applied lo a Mono 0 HR column (0.5~10 cm) previously equilibraied With buffer A. The miumn was washed withfwo bed volumes of buffer A and bound proteins were eiuted with a linear gradient 01 Tris-acetate (0-0.9 M) in buffer A. The hydroxylase was eluted as a Singleactivity peak (Fig. 2C). The peak lraclions containing 50% of the loaded hydroxylaseactivity were pooled (Table 1. step 4) and applied lo an Ultragel AcA54 column (1x47 cm) previously equilibratedwith buffer A plus 0.t M Tris-acetate. b u n d proteins were eluted with the same buffer. The hydroxylase was eluted as a single activity peak (Fig. 2D). The peak fractions containing 50 % o f the loaded hydroxylase activity were applied to a P o w 0 column (0.46~10cm) previously equilibratedwith buffer A plus 0.1 M Tns-acetate at pH 6.5. The mlumn was washed with three volumesof the equilibrating buffer and the proteirs were eluated with a linear gradientof Tris-aoetate (0.1-1.O M) in pH 6.5 Buffer A. The hydroxylasewas eluted as a single peak (Rg. ZE) and several peak fractions containingthe hydroxylaseactivity were nored at 70 "C Until required.
optimal Cataiysis. The highly purified DAOC hydroxylase requiredenernal wKG. Fez+. and 0 2 tor its activity and was stimulated by D T l and ATP. The hydroxylase was greatly nimuialed by D l 7 but only very slightly affected by ascorbate (Fig. 5). In the absence of eilher reducing agent. alow hydroxylase activlty (about 20%) was shown with Fez+and IW enzyme activity was ObseNedwith F#+. ATP WBP moderetely atimulatoIyto the enzymeat 0.1 or 0.15 mM (Fig. 5 ) . The enzymic reaction was Optimal at pH 7.0-7.4 in 15 mM MOPS buffer and 29 "C. At an Optimal pH. subslitution 01 MOPS by HEPES and1%HCI gavesimilar enzyme activities. Only one third of the meximal enzymeactivity was observed in DotaSSiumDhoSDhale buffer. An activilv difference was observed from initiation 01 the enzymic reaction bv penlciliin N, a-KG or the enzyme: the activity fmm reactlo" inltiation by penicillin N orthe enzyme was 20% higher than that by a.KG. The K, of the hydroxylase for DAOC or a-KG was analyzedat a saturating concentration01 the dtemative substrate (/.e. 300 pM 01 a-KG or DAOC). Under theoptimal reaction conditions, the Km'sof the hydroxylase for DAOC and =-KG. as determined by the Lineweaver-Burk method. were 50 and 10 pM. respectlveiy. The K. of the enzyme for Fe2+ was similarly determined as 20 pM. The, ,V of the purified hydmxylase was 0.49 pmoi DAC fomedlm~n/mgprotein.
4,932
0.03
'Determined from the hydroxylase peak fraction. ExplHyr: ExpandaseMydroxyiase. bThe ammonium sulfate traction invariably gave a higher expandaselhydmxylase anivity ratio than those from the five chromatographic steps.
Enzyme Purification. Purification of DAOC hydroxylase from S. davuligerus was performed beween 0 and 4°C. and all buffers were degassed thoroughlyprior lo use. Fresh cslls (wet weight, 400 grams) were resuspended in 15 mM MOPS, pH 7.4, in the presence 01 10 mM DTT and 10% ethanoi (buffer A) to a total volume 01 400 mi and broken by a s0nic treatment at 4 "C or below as recently described (10).
Degraded Protein: Thr Gly SerTyrThr Asp Tyr Ser Met
751
Ultrogei A d 5 4 i Eluate
Enzyme Assay. Unless othemise specified. a typical reanion mlnure of t ml for DAOC hydroxylase assay contained 0.3 pmol 01 DAOC. 0.3 pmoi of a-KG. 0.1 pmol of FeSO,, 0.25 pmol of axorbate. 1 #mol of DTT, 0.05 pmol of ATP, and 0.00005 to 0.003 unit (as defined below) of the enzymein 15 mM MOPS, pH 7.3. The enzymic reaction was iniiiated by addition 01 DAOC andconductedfor 20 mi" at 29 'C. The hydroxylase activity was determined by monitoring DAC formation at 260 nm wilh HPLC as previously described (14). DAC formation was linear with time during routine enzyme assays. One unit of the enzyme activity is defined as the amount of the hydroxylase requiredto cause formation 01 one Vmoi of DAC p e r min from D A W under the remion conditions. The specitic activity is defined as units per mg of protein. The protein content was determined by the method of Bradfod (26) using bovine serum albumin as the standard.
Native Protein: Aia Asp Thr Pro Val Pro 118 Phe Asn Leu Aia Ala Leu Arg Glu Gly AlaAsp Gin Glu Lys Phe Arg Glu Cys Val Thr Giy
15.549 5.790
DEAE-Sepharose Eluate
E
P
fP {
5093
Deacetoxycephalosporin C Hydroxylase of S. clavuligerus 'W
*p*m
l
2
O
1
A
0
A
B
Fg. 3. PuW analysis Of D A W h y d m x y h . A. SDS-PAGE and 8.Naliie-PAGE.
0.2
0.4 0.6 0.8 1.0 [Effector], mM
'
2
Fg. 5. Eneas 01 DlT, ascatvale and ATPon the hydmkylareaclivy. Paniallypunfled enqme (0.45 mulassay)was used In each elleaor anaIys8s.
15 5 I
0.1
1
0.2 0.3 0.4 0.5
(
Kav
E"c 10
-
v
c
:5 E
a
0 0 0:l 0:2 013 OI4 015 0:6
7
Relative Mobility Fig. 4. MoleQllar weighl determimlon 01 DAOC hydmxylaw. A. UHragel AW5d gel liltratron 01 panially purified enzyme and 8. SDSPAGE of highly punlied enzyme
20 40 60 80 100 120 Reaction Time (min)
FQ.6. SloiChiometrk analysns 01 DAOC hydmxylasecafalyzedmnvemon of D A W to DAC. Panially punfied enzyme fmm remmbmam E. m/i(pmvided by S. Kovacevic and J. R. Miller) was used In lhis audy. D A W and DAC were quantitatedby HPLC (11).
