©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification and Characterization of Clavaminate Synthase from Streptomyces antibioticus
A MULTIFUNCTIONAL ENZYME OF CLAVAM BIOSYNTHESIS (*)

(Received for publication, October 5, 1994; and in revised form, December 22, 1994)

James W. Janc Laura A. Egan Craig A. Townsend (§)

From the Department of Chemistry, The Johns Hopkins University, Baltimore, Maryland 21218

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Clavaminate synthase (CS), a key enzyme in the clavulanic acid biosynthetic pathway, has been purified to electrophoretic homogeneity from Streptomyces antibioticus (Tü 1718), a species that does not produce clavulanic acid. A comparison of the physical and kinetic properties of clavaminate synthase from S. antibioticus (CS3) and the two isozymes from Streptomyces clavuligerus (CS1 and CS2) has been conducted. In oxidative reactions requiring the co-substrates O(2), alpha-ketoglutaric acid, and catalytic Fe, both CS1 and CS2 catalyze three distinct transformations, the hydroxylation of deoxyguanidinoproclavaminic acid to guanidinoproclavaminic acid, and the cyclization and desaturation of proclavaminic acid to clavaminic acid. We have demonstrated that CS3 from S. antibioticus also catalyzes these three oxidations. The apparent molecular mass of CS3 from matrix-assisted laser desorption mass spectrometry is 35,839 ± 36 Da. The enzyme is a monomer in solution as determined by gel filtration chromatography. Analysis of the four possible proclavaminic acid diastereomers confirmed the absolute configuration of the substrate to be 2S,3R. Based upon N-terminal sequence comparisons among the three proteins, CS3 possesses the higher degree of homology with the CS1 isozyme from S. clavuligerus. Although previously associated solely with clavulanic acid biosynthesis, we propose these findings and recent precursor incorporation data support the view that clavaminate synthase plays a critical role in the biosynthesis of the clavam metabolites.


INTRODUCTION

Clavulanic acid (Fig. 1, clavam 1) is a clinically important beta-lactamase inhibitor produced by Streptomyces clavuligerus(1) . Clavaminate synthase (CS)^1, a non-heme iron, alpha-KG-dependent oxygenase, catalyzes three distinct transformations in the biosynthesis of 1 (Fig. 1)(2, 3) . The first of these reactions is conventional hydroxylation of deoxyguanidinoproclavaminic acid (2) to produce guanidinoproclavaminic acid (3) (4, 5) . Before CS can perform its second oxidative function, an intervening enzyme, proclavaminic acid amidino hydrolase catalyzes the hydrolysis of the guanyl moiety of 3 to produce proclavaminic acid (4)(5) . Clavaminate synthase then carries out the oxidative cyclization of 4 to produce the transient intermediate dihydroclavaminic acid (5)(6, 7) . In a third oxidative process the later is desaturated to yield clavaminic acid (6)(2, 3) . Clavaminic acid is thought to undergo oxidative deamination (8) and an unusual stereochemical inversion to yield the aldehyde (7), which is then reduced to afford 1(9) .


Figure 1: Clavulanic acid biosynthetic pathway. PAH, proclavaminic acid amidino hydrolase.



Initial isolation of CS from S. clavuligerus revealed the unexpected presence of two isozymes, CS1 and CS2(3) , whose genes (cs1 and cs2) have been cloned, sequenced(10) , and overexpressed in Escherichia coli(11) . (^2)The genes are 87% identical and the proteins differ in length by a single amino acid residue(10) . The two isozymes have the same cofactor requirements and possess nearly identical kinetic properties(3) . All genes required for the biosynthesis of 1 are believed to flank cs2(^3)in a 12-kilobase cluster (12, 13) . Surprisingly, cs1 is not present in this DNA fragment raising questions as to the role of CS1 in the biosynthesis of 1(10) .

The clavams 8, 9, 10, and 11 are members of a family of bicyclic beta-lactams structurally related to 1 (Fig. 2) and coproduced by S. clavuligerus(14, 15) . Clavams 12(16, 17) and 13(18) are isolated from an allied species, Streptomyces antibioticus, which does not produce 1. We have demonstrated that an advanced biosynthetic intermediate of clavulanic acid (1), proclavaminic acid (4), is also a precursor of clavams 8, (19) 12, and 13(20) . The common intermediacy of 4 suggests that a shared biosynthetic pathway exists to the clavams and 1, at least until the formation of 4. In view of the ``enantiomerization'' required in clavulanic acid formation(8) , we have postulated that the branch point of these two pathways may lie beyond clavaminic acid (6)(19, 20) . Accordingly, we have proposed that CS may be present in S. antibioticus, the organism responsible for the production of 12 and 13 alone, and that this multifunctional enzyme may play a role in the biosynthesis of the clavams(20, 21) .


Figure 2: Structures of the clavam metabolites.



In this study we report the purification to homogeneity and thorough characterization of clavaminate synthase (CS3) from S. antibioticus. A comparison of the physical and kinetic properties of CS3 from S. antibioticus to the two isozymes from S. clavuligerus has been completed and has demonstrated that many common features exist among the three enzymes.


EXPERIMENTAL PROCEDURES

Materials

Yeast extract and malt extract were obtained from Difco, D-glucose from J. T. Baker Inc., D-mannitol and phthalic o-dicarboxaldehyde from Aldrich, and ammonium sulfate and DTT from Fluka Chemie AG (Buchs, Switzerland). Whole soybeans (Arrowhead Mills, Hereford, TX) were purchased from a local health food store. Leupeptin, DL-methionine, benzamidine hydrochloride, EDTA (disodium salt), benzethonium hydroxide (1.0 M solution in methanol), PMSF, soybean trypsin inhibitor, alpha-KG (free acid), sodium ascorbate, Me(2)SO-based ninhydrin reagent, BSA, catalase (bovine liver), PEP (monocyclohexylammonium salt), GTP (lithium salt), coenzyme A (lithium salt), Tris, MOPS (free acid), ADP (disodium salt), NADH (disodium salt), pyruvate kinase (rabbit muscle), L-lactate dehydrogenase (porcine muscle), glutamate-pyruvate transaminase (porcine heart), succinic thiokinase (porcine heart), DEAE-Sepharose CL-6B, and Sephadex G-75 Superfine were from Sigma. [1-^14C]alpha-KG was purchased from DuPont-NEN. All other chemicals were reagent grade and used without further purification. Optically pure and racemic proclavaminic acid and the racemic erythro isomers were synthesized and correlated to L-glutamic acid by methods described elsewhere(7, 22) . Concentrations of proclavaminic acid solutions were determined with a ninhydrin assay using beta-alanine as the standard(23) . Imidazole reagent used to assay the conversion of proclavaminic acid to clavaminic acid consisted of a 3 M aqueous solution of imidazole adjusted with HCl to pH 6.8(24) .

Growth of S. antibioticus

Seed medium (pH 7.3) contained, per liter, DL-methionine (0.4 g), D-glucose (4.0 g), yeast extract (4.0 g), and malt extract (10 g). Fermentation medium contained 20 g of freshly ground soy beans and 30 g of D-mannitol per liter (pH 7.2). Seed flasks were inoculated from a spore suspension of S. antibioticus (Tü 1718) prepared as described by Wanning(25) . Seed flasks were incubated with shaking at 300 rpm, 27 °C, for 48 h. Erlenmeyer flasks each containing 100 ml of fermentation medium were inoculated with 1 ml of the 48-h seed medium and shaken at 300 rpm, 27 °C, for 72 h. Fermentation broth was assayed for clavam production by derivatization with imidazole reagent and measurement of the absorbance of products at 315 nm(3, 24) . Clavam production was calculated assuming = 26,900 M cm(24) . Cells were harvested by centrifugation during the production phase (72 h, 170 g cells/liter), frozen in liquid N(2), and stored at 78 °C.

Protein Purification

All steps were performed at 4 °C. General enzyme buffer (GEB) refers to 50 mM Tris (pH 7.5 at 25 °C), 1 mM DTT, 20 µM EDTA, 0.2 mM PMSF, 1.0 mM benzamidine, and 5% glycerol. Frozen S. antibioticus cells (50 g) were suspended in 100 ml of sonication buffer containing: 50 mM Tris (pH 7.5 at 25 °C), 1 mM EDTA, 2 mM DTT, 1 mM benzamidine, 1 µM leupeptin, 5 mg/liter soybean trypsin inhibitor, 1 mM PMSF, and 10% glycerol. Cells were lysed by sonication (Heat Systems-Ultrasonics model 225R, Plainview, NY), and cellular debris was removed by centrifugation. One-fifth volume of a 5% (w/v) streptomycin sulfate solution was added to the cell-free extract to precipitate the nucleic acids which were removed by centrifugation. To the supernatant was added solid ammonium sulfate over 20 min to reach 40% of saturation and precipitated protein was removed by centrifugation. The supernatant was brought to 70% of saturation by the addition of ammonium sulfate and the insoluble protein was pelleted by centrifugation. The resulting pellet was dissolved in a minimum volume of GEB and dialyzed (Spectrum Medicinal Industries, Los Angeles, CA; Spectra/Por 10,000 molecular weight cut off) against 2 liters of GEB for 18 h. The desalted protein solution (22 ml) was diluted to 60 ml and loaded onto a DEAE-Sepharose CL-6B column (2.5 times 15 cm, 74-ml bed volume) previously equilibrated in GEB. The column was eluted with one bed volume of GEB followed by a linear gradient of NaCl from 0 to 350 mM in a total volume of 800 ml. CS3 eluted at 150 mM NaCl. Active fractions were concentrated in an Amicon ultrafiltration apparatus (Beverly, MA; 50 ml cell, PM 10 membrane), loaded onto a Sephadex G-75 Superfine column (1.5 times 95 cm, 168-ml bed volume), and eluted with GEB. Fractions containing CS3 activity were concentrated by ultrafiltration using a Centricon-10 (Amicon) during which time the G-75 buffer was exchanged with 50 mM MOPS, pH 7.0, 20 µM EDTA, 2 mM DTT, and 50% glycerol. The protein was stored at -80 °C where it is stable for at least 1 year.

Assays

Assays for specific activity during the purification procedure were carried out according to the protocol of Salowe et al.(3) . The standard assay used elsewhere in this report to monitor the conversion of proclavaminic acid to clavaminic acid followed Salowe's procedure(3) . With the exception of the experiments examining the stereospecificity of the enzyme, all assays used rac-threo-proclavaminic acid. In the experiments with alpha-KG as a variable substrate, an additional correction was applied, using = 17.9 M cm, experimentally determined for this assay to compensate for the absorbance attributed to alpha-KG. In the experiments with Fe as a variable substrate, = 6.1 mM cm was used to correct for the FebulletEDTA complex. The concentrations of alpha-KG stock solutions were calibrated enzymatically by using glutamate-pyruvate transaminase and L-lactate dehydrogenase as coupling enzymes and following NADH oxidation. The concentrations of stock iron solutions were determined spectrophotometrically using Ferrozine(26) . In kinetic experiments substrate consumption was always less than 10% for proclavaminic acid and less than 20% for alpha-KG. Kinetic data were fitted to the appropriate equations with the FORTRAN programs of Cleland to obtain the desired kinetic parameters(27) :

In and , V represents the maximum velocity, A is the concentration of the variable substrate, K(a) is the Michaelis constant for A, and K(1) and K(2) are saturation constants for a substrate which binds twice to the enzyme with no intervening irreversible steps.

In radioactive assays measuring carbon dioxide formation the assay components were identical to those in the standard assay except for the substitution of [1-^14C]alpha-KG diluted with radioinactive material to a specific activity of 0.34 µCi/µmol. Incubations were performed in 8-ml glass vials sealed with rubber septa and fitted with plastic center wells (Kontes, Vineland, NJ). Each well contained a piece of filter paper impregnated with 25 µl of 1 M benzethonium hydroxide in methanol. The assays were initiated by syringe addition of enzyme and terminated with 50 µl of 50% trifluoroacetic acid. After a 1-h incubation at 37 °C, the center wells were transferred to scintillation vials for counting in 20 ml of Opti-Fluor O mixture (Packard, Downers Grove, IL). Corrections were applied for non-enzymatic decarboxylation of alpha-KG. The production of succinate was measured with a coupled enzyme assay as described by Beutler(28) .

The assay for the conversion of 2 to 3 was performed using reverse phase HPLC. The stationary phase was Versapack, C-18 (10µm, 0.46 times 25 cm) and the mobile phase was 100% H(2)O CS3 (21.6 µg) was incubated in 50 mM MOPS, pH 7. 0, 0.5 mM DTT, 0.1 mM ascorbate, 1.0 mM alpha-KG, 25 µM Fe, and 1.2 mM2 at 30 °C. After 30 min of incubation the reaction mixture was analyzed by HPLC. The product, 3, was identified by coelution with an authentic sample.

The assay used to monitor the conversion of 4 to 6 involved product derivatization with phthalic o-dicarboxaldehyde (OPA) followed by HPLC analysis(29, 30) . An OPA derivatization mixture (45 mM OPA in 0.54 M sodium borate, pH 9.5, containing 1% beta-mercaptoethanol) was prepared and placed on ice in the dark. CS3 was incubated with 4 under standard conditions at 25 °C as above. Aliquots (15µl) were removed after 30 min, mixed with 15µl of OPA derivatization mixture for 30 s, and analyzed by HPLC using a Spherisorb octadecyl sulfate 2 C-18 column (5 µm, 0.46 times 25 cm). Derivatized products were monitored at 340 nm.

Other Methods

UV-visible spectrophotometry employed a Beckman DU 70 Spectrophotometer (Fullerton, CA). Protein assays were performed by the method of Bradford (31) using BSA as a standard. SDS-PAGE used the buffer system of Laemmli (32) in a Hoefer SE 400 Slab Gel Electrophoresis Unit (San Francisco, CA). Radioactive measurements were made with a Beckman LS 5801 liquid scintillation counter using Opti-Fluor O scintillation mixture. Protein HPLC to determine the molecular weights of native proteins was performed using a Waters 625 HPLC (Milford, MA) system equipped with PEEK tubing and a Pharmacia Superose 12 HR 10/30 column (Uppsala, Sweden). The column was equilibrated in 50 mM Tris (pH 7.5 at 25 °C) and 150 mM KCl. The following protein standards were used to generate the calibration curve: beta-amylase (M(r) 200,000), alcohol dehydrogenase (M(r) 150,000), BSA (M(r) 66,000), carbonic anhydrase (M(r) 29,000), and cytochrome c (M(r) 12,400). For N-terminal sequence, amino acid composition, and mass spectral analysis, a sample of CS3 was further purified by HPLC using a Vydac C-4 reverse phase column (0.5 times 15 cm). The mobile phase consisted of a gradient of 100% water containing 0.1% trifluoroacetic acid to 100% acetonitrile containing 0.1% trifluoroacetic acid, and CS3 eluted at 53% acetonitrile. Purified samples of CS3 were carboxymethylated and then hydrolyzed in 6 M HCl. Amino acids were derivatized with phenyl isothiocyanate prior to analysis on Waters PICO-TAG amino acid analysis system. N-terminal sequencing was performed using an Applied Biosystems 470A gas-phase protein sequencer (Foster City, CA). Protein mass spectrometry was performed using the method of matrix-assisted laser desorption(33) .


RESULTS

Protein Purification and Physical Characterization

A newly isolated clavaminate synthase (CS3) has been purified to electrophoretic homogeneity from S. antibioticus. The purification of CS3, based largely on the protocol of Salowe, employed ammonium sulfate fractionation, anion exchange chromatography, and gel filtration chromatography, as summarized in Table 1(3) . A total of 3.49 mg of CS3 was obtained from 50 g of S. antibioticus cells, while 50 g of S. clavuligerus cells yielded 7.0 mg of a mixture of CS1 and CS2(3) .



With homogeneous CS3 in hand, we set out to determine whether CS3 performed each of the three transformations catalyzed by the S. clavuligerus isozymes. Following incubation of CS3 with deoxyguanidinoproclavaminic acid (2), HPLC analysis of the reaction mixture confirmed the presence of guanidinoproclavaminic acid (3) as evidenced by coelution with an authentic sample. The conversion of proclavaminic acid (4) to clavaminic acid (6) catalyzed by CS3 was monitored in two ways. Following CS3 incubation, addition of imidazole to the reaction mixture resulted in derivatization of 6 and production of an alpha,beta-unsaturated acyl imidazole adduct whose chromophore was monitored at 312 nm. The oxidative cyclization/desaturation reactions were further observed by derivatizing the reaction mixture with OPA and analyzing the derivatized products by reverse phase HPLC. By this method we were also able to detect low levels of dihydroclavaminic acid (5).

N-terminal sequence analysis was carried out and comparisons were made among all the CS enzymes (Table 2). This limited comparison revealed that the higher degree of homology exists between CS3 and CS1, the isozyme whose gene is not located in the clavulanic acid biosynthetic operon. The amino acid composition of CS3 was determined and the results are compiled with those translated from the gene sequences of CS1 and 2 (Table 3)(^4)(10) . The amino acid composition of CS3 is similar to those of CS1 and CS2 and is typical of a globular protein, with some notable exceptions. Amino acids utilizing codons rich in A and T, for example phenylalanine and lysine, were present in unusually low amounts. Conversely, amino acids such as arginine and proline, whose codons are primarily G and C, appear more frequently in CS3 than in most globular proteins(34) . This deviation from the predicted amino acid distribution can be rationalized by the fact that Streptomyces DNA has a high (74%) GC content (35) .





The UV/visible spectrum of CS3 was recorded from 240-600 nm and a single, broad absorbance maximum was observed at 280 nm. The lack of any longer wavelength absorbances in the spectrum of CS3 suggests that there are no tightly bound cofactors present. The molecular weight of CS3 has been estimated by several methods and these results were compared with those previously reported for CS1 and 2 (Table 4). The mass of CS3 was determined using matrix-assisted laser desorption (MALD) mass spectrometry, and a value of 35,839 ± 36 Da was obtained(33) . Analysis by SDS-PAGE revealed that CS3 migration is consistent with the molecular weight determined by MALD mass spectrometry (Fig. 3). This behavior distinguishes CS3 from its S. clavuligerus counterparts whose apparent molecular weights by SDS-PAGE differ significantly (3) from both the molecular weights translated from the gene sequence and those determined by MALD mass spectrometry (Table 4). CS3 behaves as a monomer when analyzed by Superose 12 fast protein liquid chromatography under non-denaturing conditions consistent with the results obtained for CS1 and CS2.




Figure 3: SDS-PAGE comparison of clavaminate synthases. Ten percent polyacrylamide gel was stained with Coomassie Brilliant Blue, and recombinant CS1 and CS2 were used as standards.



Stereospecificity and Reaction Stoichiometry

Of the four possible diastereomers of 4, it has been shown that the 2S,3R stereoisomer is the true substrate for CS1 and CS2(3, 36) . In order to independently confirm that the natural substrate of CS3 is identical to that of CS1 and CS2, CS3 was incubated with all possible stereoisomers of 4. Initial incubation of CS3 with rac-erythro4 (2S,3S and 2R,3R) showed that CS3 failed to convert these substrates into 6, as monitored by the imidazole assay. This led to an individual examination by HPLC of each of the optically pure threo enantiomers of 4, which confirmed the true substrate was indeed the 2S,3R enantiomer.

The transformation of 4 to 6 occurs in a stepwise fashion in which each of the two oxidative half reactions results in the decarboxylation of alpha-KG to form succinate and CO(2). In parallel reactions utilizing [1-^14C]alpha-KG and radioinactive alpha-KG, the evolution of ^14CO(2) and production of 6 were monitored. Two moles of CO(2) were evolved for each mole of 6 produced (Fig. 4). Qualitatively, the formation of succinate was confirmed using the succinic thiokinase coupled assay of Beutler(28) .


Figure 4: Comparison of CO(2) evolution with clavaminate formation. Parallel reactions under standard conditions containing 1.0 mM alpha-KG were assayed for clavaminate formation by spectrophotometric assay, circle, or for CO(2) formation by radiochemical assay, bullet.



Enzyme Stability

Salowe and co-workers have reported that CS from S. clavuligerus undergoes rapid self-inactivation when incubated in a reaction mixture containing all required cofactors but devoid of 4(3) . It was shown that inclusion of catalase in assay mixtures afforded protection to the enzyme. These findings suggest that in the absence of 4, the putative iron-oxygen intermediate can inflict oxidative damage or release hydrogen peroxide leading to enzyme inactivation.

We sought to determine whether CS3 was susceptible to the same oxidative self-inactivation observed for CS1 and CS2. CS3 is stable in a buffered solution at 25 °C in the absence of 4 and all cofactors except endogenous O(2) (t = 19 h) (Fig. 5). However, when CS3 was incubated under parallel conditions with Fe, alpha-KG, and O(2), rapid loss of catalytic activity resulted (t = 52 s). Addition of catalase (0.1 mg/ml) provided partial protection to CS3 from oxidative damage (t = 46 min). The role of catalase appears to be specific as substitution by BSA for catalase at the same concentration failed to provide similar protection (t = 72 s).


Figure 5: Auto-inactivation of CS3 in the absence of proclavaminic acid. CS3 was incubated in 50 mM MOPS buffer, pH 7.0, containing 0.5 mM DTT at 25 °C alone or with various additives: bullet, no additions; box, 10 µM Fe, 100 µM ascorbate, 1.0 mM alpha-KG; , same as box, plus 0.1 mg/ml catalase.



The rapid inactivation of CS3 is consistent with data obtained for CS1 and CS2 and further supports the suggestion that the mechanism of oxygen activation is shared among all three enzymes. The observation that catalase offers protection to the enzyme implies the formation of hydrogen peroxide is occurring. However, the inability of catalase to afford complete protection to CS3 indicates that diffusable hydrogen peroxide cannot be the sole causative agent responsible for oxidative damage leading to inactivation. The production of peroxide in the incomplete reaction mixture implies that CS3 can catalyze a partial reaction in which oxidative decarboxylation of alpha-KG results in the production of an activated iron-oxygen species. When 4 is unavailable for oxidative reaction, this high energy oxygen species may be redirected toward residues at or near the active site resulting in self-inactivation.

Steady State Kinetics

We have determined the relevant kinetic parameters for Fe, alpha-KG, and 4 in the reaction catalyzed by CS3. These same parameters were previously determined for a mixture of CS1 and CS2(3) . A comparison of the kinetic parameters of the CS enzymes is presented in Table 5, which illustrates that there is no remarkable deviation in the Michaelis constants observed for CS3 with those measured previously for CS1 and CS2.



A stepwise mechanism has been proposed for CS1 and CS2 involving two discrete oxidative half reactions occurring at the same active site. In the first, 4 is oxidatively cyclized to 5, while consuming alpha-KG and O(2) and producing CO(2), succinate, and H(2)O. In the second half reaction, 5 undergoes desaturation also consuming alpha-KG and O(2) and releasing CO(2), succinate, and H(2)O. In the CS3 catalyzed conversion of 4 to 6 we sought to examine the dependence of velocity upon the concentration of the substrate, alpha-KG. A linear double-reciprocal plot was obtained when alpha-KG was supplied as the variable substrate (Fig. 6). This result is consistent with the stepwise mechanism proposed for CS1 and CS2 in which the binding of alpha-KG is separated by an irreversible step, in this case the release of succinate and CO(2). For a case in which the same substrate binds twice and each binding event is not separated by an irreversible step, theory predicts a parabolic double-reciprocal plot (37) . The velocity dependence upon substrate concentration in this case is described by , however attempts to fit our data to did not yield a satisfactory result.


Figure 6: Double-reciprocal plot of the dependence of initial velocity on alpha-KG concentration. The concentration of alpha-KG was varied from 0.05 to 1.25 mM. The concentrations of the nonvariable substrates were 1.5 mM for rac-proclavaminic acid and 50 µM for Fe. Velocity measurements were performed in duplicate at 0.05, 0.113, and 1.25 mM alpha-KG. The line shown represents the fit of the data to .




DISCUSSION

We have purified clavaminate synthase (CS3) from S. antibioticus, an organism which does not produce clavulanic acid (1). This multifunctional enzyme performs the identical three oxidative reactions catalyzed by the S. clavuligerus isozymes in the biosynthesis of 1. All three synthases require the same cofactors, namely Fe, alpha-KG, and O(2) and have closely similar physical and kinetic properties ( Table 4and Table 5).

N-terminal sequence analysis demonstrated that CS3 shows greater homology to the CS1 isozyme than to CS2.^4 While this comparison is made with only the first 20 amino acids of each protein, it should be borne in mind that the genes for CS1 and CS2 show very high levels of identity in their central regions with the greatest divergence occurring at the N and C termini(10) . Therefore, the similarities and differences seen in this sample may well be more meaningful than its limited size might initially suggest. Interestingly, the gene coding for CS1 is not present in the clavulanic acid operon of S. clavuligerus. This organism coproduces several clavam metabolites along with 1. It is possible that the CS1 gene resides in a genomic region dedicated to the production of clavams in S. clavuligerus. S. antibioticus, however, produces CS yet fails to produce 1. We propose that CS3 is required for clavam biosynthesis and, therefore, might be expected to be more homologous to the isozyme CS1 of S. clavuligerus.

Previous work has shown that proclavaminic acid (4) is an intermediate in clavam and clavulanic acid biosynthesis(19, 20) . We have demonstrated the ability of CS3 to hydroxylate deoxyguanidinoproclavaminic acid (2) to guanidinoproclavaminic acid (3), clearly implicating CS3 in the production of 4 in S. antibioticus. Further, proclavaminic amidino hydrolase activity has been detected in S. antibioticus, which provides a means of converting 3 to 4(21) . Additionally, CS3 possesses the ability to convert 4 to 6, a reaction known to occur in the biosynthesis of 1. If 6 is the in vivo product of the reaction of 4 with CS3, then the likelihood is increased that 6 plays a role in the biosynthesis of the clavams as well. Based on the stereochemical similarities that exist between 6 and the clavams, the intermediacy of 6 in their biosyntheses can be readily envisioned.

An extensive, shared biosynthetic pathway between the clavams and 1, including the intermediacy of 6, has been previously proposed (20) and the purification of CS3 from S. antibioticus strengthens this notion (Fig. 7). The partitioning of the two pathways is suggested to occur after the proposed intermediacy of clavaminic acid (6). An oxidative deamination occurs without decarboxylation but with stereochemical ring inversion to give the aldehyde, 7(8, 9) known to be the penultimate precursor of clavulanic acid (1). The same or similar intermediate, through decarboxylation but without inversion, has been proposed to afford the aldehyde 14. This intermediate is thought to be common to the individual members of the clavam family; for example, aldol condensation and further reactions can be envisioned to provide 12, while simple reduction gives 13. This proposal emphasizes the critical role CS3 plays in the biosynthesis of clavams 12 and 13 in S. antibioticus and relates to the open question of the location and organization of the clavam biosynthetic genes in S. clavuligerus.


Figure 7: Biosynthetic parallels among clavulanic acid and the clavams.




FOOTNOTES

*
This work was funded in part by National Institutes of Health Grant AI 14937 (to C. A. T.) and National Institutes of Health Postdoctoral Fellowship GM 15174 (to J. W. J.). Funding to acquire the major analytical instrumentation was obtained from the National Institutes of Health and the National Science Foundation (NMR: RR 04794, RR 01934, and PCM 83-03176; MS: RR 02318). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 410-516-7444; Fax: 410-516-8420.

(^1)
The abbreviations used are: CS, clavaminate synthase; BSA, bovine serum albumin; DTT, dithiothreitol; alpha-KG, alpha-ketoglutaric acid (2-oxopentanedioic acid); MOPS, 3-(N-morpholino)propanesulfonic acid; PEP, phosphoenolpyruvate; PMSF, phenylmethylsulfonyl fluoride; OPA, phthalic o-dicarboxaldehyde; PAGE, polyacrylamide gel electrophoresis; MALD, matrix-assisted laser desorption; HPLC, high performance liquid chromatography.

(^2)
Busby, R. W. Chang, M. D.-T., Busby, R. C., Wimp, J., and Townsend, C. A.(1995) J. Biol. Chem.270, 4262-4269.

(^3)
T.-K. Wu, T. A. Houston, R. W. Busby, D. B. McIlwaine, L. A. Egan, and C. A. Townsend, submitted for publication.

(^4)
It is possible to align the sequences differently and increase the apparent degree of homology between CS2 and CS3.


ACKNOWLEDGEMENTS

We are grateful to Professor Hans Zähner of the University of Tübingen for generously providing a slant of S. antibioticus (Tü 1718). We thank Dr. W.-S. Liu of the Protein Peptide Facility, The Johns Hopkins School of Medicine, for providing the N-terminal sequence and amino acid composition analyses of clavaminate synthase. Mass spectral measurements were carried out by Dr. A. S. Woods at the Middle Atlantic Mass Spectrometry Facility at The Johns Hopkins University, a National Science Foundation Shared Instrumentation Facility. We are grateful to Dr. W. J. Krol and Dr. T. A. Houston for preparation of some of the substrates used in this work and R. W. Busby for providing samples of recombinant clavaminate synthase.


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