Crystal Structure and Mechanistic Implications of N2-(2-Carboxyethyl)arginine Synthase, the First Enzyme in the Clavulanic Acid Biosynthesis Pathway

Abstract

The initial step in the biosynthesis of the clinically important β-lactamase inhibitor clavulanic acid involves condensation of two primary metabolites, d-glyceraldehyde 3-phosphate and l-arginine, to give N2-(2-carboxyethyl)arginine, a β-amino acid. This unusual N-C bond forming reaction is catalyzed by the thiamin diphosphate (ThP2)-dependent enzyme N2-(2-carboxyethyl)arginine synthase. Here we report the crystal structure of N2-(2-carboxyethyl)arginine synthase, complexed with ThP2 and Mg2+, to 2.35-Å resolution. The structure was solved in two space groups, P212121 and P21212. In both, the enzyme is observed in a tetrameric form, composed of a dimer of two more tightly associated dimers, consistent with both mass spectrometric and gel filtration chromatography studies. Both ThP2 and Mg2+ cofactors are present at the active site, with ThP2 in a “V” conformation as in related enzymes. A sulfate anion is observed in the active site of the enzyme in a location proposed as a binding site for the phosphate group of the d-glyceraldehyde 3-phosphate substrate. The mechanistic implications of the active site arrangement are discussed, including the potential role of the aminopyrimidine ring of the ThP2. The structure will form a basis for future mechanistic and structural studies, as well as engineering aimed at production of alternative β-amino acids. The initial step in the biosynthesis of the clinically important β-lactamase inhibitor clavulanic acid involves condensation of two primary metabolites, d-glyceraldehyde 3-phosphate and l-arginine, to give N2-(2-carboxyethyl)arginine, a β-amino acid. This unusual N-C bond forming reaction is catalyzed by the thiamin diphosphate (ThP2)-dependent enzyme N2-(2-carboxyethyl)arginine synthase. Here we report the crystal structure of N2-(2-carboxyethyl)arginine synthase, complexed with ThP2 and Mg2+, to 2.35-Å resolution. The structure was solved in two space groups, P212121 and P21212. In both, the enzyme is observed in a tetrameric form, composed of a dimer of two more tightly associated dimers, consistent with both mass spectrometric and gel filtration chromatography studies. Both ThP2 and Mg2+ cofactors are present at the active site, with ThP2 in a “V” conformation as in related enzymes. A sulfate anion is observed in the active site of the enzyme in a location proposed as a binding site for the phosphate group of the d-glyceraldehyde 3-phosphate substrate. The mechanistic implications of the active site arrangement are discussed, including the potential role of the aminopyrimidine ring of the ThP2. The structure will form a basis for future mechanistic and structural studies, as well as engineering aimed at production of alternative β-amino acids. An important mechanism of bacterial resistance to β-lactam antibiotics involves β-lactamase-catalyzed hydrolysis of the β-lactam ring, resulting in biologically inactive products (1Matagne A. Dubus A. Galleni M. Frère J.-M. Nat. Prod. Rep. 1999; 16: 1-18Crossref PubMed Scopus (182) Google Scholar). Clavulanic acid (Fig. 1), one of many β-lactams produced by Streptomyces clavuligerus, is the most widely used inhibitor of the serine β-lactamases, and is clinically administered in combination with the penicillin amoxycillin (2Jensen S.E. Paradkar A.S. Antonie Leeuwenhoek Int. J. Gen. Mol. Microbiol. 1999; 75: 125-133Crossref PubMed Scopus (67) Google Scholar, 3Baggaley K.H. Brown A.G. Schofield C.J. Nat. Prod. Rep. 1997; 14: 309-333Crossref PubMed Scopus (137) Google Scholar). Clavulanic acid is itself a bicyclic β-lactam, and reacts with the β-lactamases to form stable acyl-enzyme complexes (4Brown R.P. Aplin R.T. Schofield C.J. Biochemistry. 1996; 35: 12421-12432Crossref PubMed Scopus (101) Google Scholar, 5Knowles J.R. Acc. Chem. Res. 1985; 18: 97-104Crossref Scopus (222) Google Scholar). A commercially viable synthesis of clavulanic acid is rendered difficult by the high lability and density of functionalization around the bicyclic core (3Baggaley K.H. Brown A.G. Schofield C.J. Nat. Prod. Rep. 1997; 14: 309-333Crossref PubMed Scopus (137) Google Scholar, 6Bentley P.H. Brooks G. Gilpin M.L. Hunt E. Tetrahedron Lett. 1979; 20: 1889-1890Crossref Scopus (10) Google Scholar). Consequently clavulanic acid is produced by fermentation and there is considerable interest in its biosynthetic pathway. A total of 18 genes from the genome of S. clavuligerus have thus far been implicated in clavulanic acid biosynthesis (7Mellado E. Lorenzana L.M. Rodriguez-Saiz M. Diez B. Liras P. Barredo J.L. Microbiol. SGM. 2002; 148: 1427-1438Crossref PubMed Scopus (56) Google Scholar, 8Li R.F. Khaleeli N. Townsend C.A. J. 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Bacteriol. 1995; 177: 3714-3720Crossref PubMed Google Scholar, 18Elson S.W. Baggaley K.H. Davidson M. Fulston M. Nicholson N.H. Risbridger G.D. J. Chem. Soc. Chem. Commun. 1993; 15: 1212-1214Crossref Google Scholar, 19Aidoo K.A. Wong A. Alexander D.C. Rittamer R.A. Jensen S. Gene (Amst.). 1994; 147: 41-46Crossref PubMed Scopus (58) Google Scholar). Furthermore, the enzymes β-lactam synthetase, proclavaminate amidino hydrolase, and clavaminic acid synthase, products of orfs3, -4, and -5, respectively, have been structurally characterized (20Miller M.T. Bachmann B.O. Townsend C.A. Rosenzweig A.C. Nat. Struct. Biol. 2001; 8: 684-689Crossref PubMed Scopus (58) Google Scholar, 21Elkins J.M. Clifton I.J. Hernandez H. Doan L.X. Robinson C.V. Schofield C.J. Hewitson K.S. Biochem. J. 2002; 366: 423-434Crossref PubMed Google Scholar, 22Zhang Z. Ren J. Stammers D.K. Baldwin J.E. Harlos K. Schofield C.J. Nat. Struct. Biol. 2000; 7: 127-133Crossref PubMed Scopus (252) Google Scholar). open reading frame N2-(2-carboxyethyl)arginine synthase thiamin diphosphate yeast pyruvate decarboxylase electrospray ionization mass spectrometry Zymomonas mobilis pyruvate decarboxylase. N2-(2-Carboxyethyl)arginine synthase (CEAS) is the enzyme product of the first gene in the clavulanic acid cluster, orf2. It catalyzes the first step in the clavulanic acid pathway, the condensation of l-arginine and a three-carbon precursor to give N2-(2-carboxyethyl)arginine (Fig. 1). On the basis of turnover experiments with a variety of potential three carbon precursors the best substrate identified was d-glyceraldehyde 3-P (12Khaleeli N. Li R. Townsend C.A. J. Am. Chem. Soc. 1999; 121: 9223-9224Crossref Scopus (82) Google Scholar). Sequence analysis indicated the presence of a conserved thiamin diphosphate (ThP2) binding fold, and indeed, CEAS catalysis has been shown to be dependent on ThP2 (12Khaleeli N. Li R. Townsend C.A. J. Am. Chem. Soc. 1999; 121: 9223-9224Crossref Scopus (82) Google Scholar). ThP2-dependent enzymes generally employ the cofactor both to effect nucleophilic attack at a carbonyl group and to stabilize the carbanion subsequently formed by proton transfer or decarboxylation. The reactions catalyzed by ThP2 enzymes may be subclassified as either non-oxidative, such as those of yeast pyruvate decarboxylase (PDC) (23Jordan F. Nat. Prod. Rep. 2003; 20: 184-201Crossref PubMed Scopus (201) Google Scholar) and benzoylformate decarboxylase (24Hasson M.S. Muscate A. McLeish M.J. Polovnikova L.S. Gerlt J.A. Kenyon G.L. Petsko G.A. Ringe D. Biochemistry. 1998; 37: 9918-9930Crossref PubMed Scopus (164) Google Scholar), or oxidative, such as those of pyruvate oxidase (25Muller Y.A. Schulz G.E. Science. 1993; 259: 965-967Crossref PubMed Scopus (215) Google Scholar, 26Bertagnolli B.L. Hager L.P. J. Biol. Chem. 1991; 266: 10168-10173Abstract Full Text PDF PubMed Google Scholar). More recently, several ThP2 enzymes that catalyze atypical reactions have been identified, including CEAS and 2-hydroxyphytanoylcoenzyme A lyase, an enzyme involved in the degradation of phytanic acid that is derived from the side chain of chlorophyll (27Foulon V. Antonenkov V.D. Croes K. Waelkens E. Mannaerts G.P. Van Veldhoven P.P. Casteels M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10039-10044Crossref PubMed Scopus (94) Google Scholar). In addition to its key role in clavam biosynthesis, CEAS is of mechanistic interest due to the unprecedented manner in which a ThP2-dependent enzyme is used in a N-C bond forming reaction, proposed to proceed via an α,β-unsaturated acyl-ThP2 intermediate. Here we report the crystal structure of CEAS complexed with ThP2 and Mg2+, and discuss the mechanistic implications arising. Protein Expression and Purification—The ceas gene was PCR amplified and subcloned directly as an NdeI/BamHI fragment into the pET24a(+) vector (Novagen). Primers used were: forward, 5′-acgctcatatgtcccgtgta-3′; reverse, 5′-ggacatgagcggtctcccgactccgtcagtacc-3′. Following amplification, the integrity of ceas was confirmed by DNA sequencing. The ceas/pET24a(+) construct was transformed into Escherichia coli BL21(DE3) and grown at 37 °C in 2TY medium containing 30 μg/ml kanamycin. Protein production was induced at an A600 of 0.8 by addition of isopropyl-β-d-thiogalactopyranoside to a final concentration of 1.0 mm, following reduction of the temperature to 30 °C. CEAS was purified by sequential use of Q-Sepharose and phenyl-Resource chromatography. Size exclusion chromatography (Superdex S200) was used as a final step to yield CEAS of >95% purity by SDS-PAGE analysis. Electrospray ionization mass spectrometry (ESI-MS) revealed that the mass of the isolated CEAS was consistent with loss of N-terminal methionine from the predicted amino acid sequence (observed, 60,776 Da; calculated without N-terminal methionine, 60,776 Da). N-terminal amino acid sequence analysis (observed, SRVSTAPSGK) confirmed the identity of the protein and the loss of the N-terminal methionine. The activity of purified CEAS was confirmed by high performance liquid chromatography assay and comparison with known synthetic standards. Selenomethionine-substituted Protein Expression—Selenomethionine-substituted CEAS was produced using a metabolic inhibition protocol and LeMaster media supplemented with 50 mg/liter l-selenomethionine. Selenomethionine incorporation was >95% by ESI-MS. Native Molecular Weight Determination—The “native” molecular weight of CEAS was determined by size exclusion chromatography (Superdex 200 HR). Calibration was carried out using cytochrome c (12 kDa), chymotrypsin (25 kDa), ovalbumin (43 kDa), bovine serum albumin (67 kDa), aldolase (158 kDa), and blue dextran (2000 kDa) (Gel Filtration Calibration kit, Amersham Biosciences). An elution volume parameter (Kav) was calculated for each of the calibration proteins and a calibration curve was constructed. By calculating Kav for CEAS the native molecular weight was established. Crystallization—Crystallization conditions were initially sought by the vapor diffusion method, using commercially available crystallization screens. Hanging drops of 2 μl of protein (10 mg/ml), containing a 3-fold excess of thiamin diphosphate, and 2 μl of well solution were suspended at 17 °C over a well solution containing 1.6 m ammonium sulfate and 0.1 m HEPES, pH 7.4. Under these conditions, crystals of two different morphologies grew over a period of 2 weeks. The crystals to two different space groups, the of the crystals was a of the space The of the crystals to space group with a c The of the crystals to space group with a c and were by into liquid and were at using a was by sequential transfer into of m ammonium containing and from crystals was on at the using a were on and of the The were using A.G. Biol. 1999; PubMed Scopus Google Scholar) and the of Biol. 1994; PubMed Scopus Google Scholar). The crystal used for the structure solution to the P212121 space Biol. 2002; Scopus Google Scholar), were to the N-terminal methionine that was present by N-terminal and ESI-MS. and density were using P. R.W. J.S. J. M. N.S. R.J. L.M. G.L. Biol. 1998; PubMed Scopus Google Scholar). The a of of over the to which to following density and are shown in and c in the in the of in the in the of total in the in the in the in the in the in the in the in the in the in the bond in for side and in the in the in for side and in a initial was using the T.A. S.W. M. A. 1991; 47: PubMed Scopus Google Scholar). of the using a was using of by the to of the were for of of using A. K.S. E.J. Biol. 1999; PubMed Scopus Google Scholar), including and the addition of and ThP2 the to and the to were used The for in the for which the density was In total the final protein out of a of to R.A. J.M. J. 1993; Google Scholar) there were The 2.35-Å native was solved by using of the to and the to The crystal form was solved by molecular with the 2.35-Å structure using were of was using were with J. 1991; Google Scholar), R.M. J. Mol. 1997; 15: PubMed Scopus Google Scholar), and 1997; PubMed Scopus Google Scholar). CEAS crystals were in the presence of using ammonium sulfate as crystals that were in the of ThP2 a variety of The crystals grew in two and The of the crystals were of the The crystals to two different space groups, P212121 and with Both space were observed for the crystals as the P212121 space group has been observed for the structure of CEAS has been determined to 2.35-Å in the P212121 crystal form using the and the form was subsequently solved to by molecular In both crystal a tetrameric arrangement of CEAS was observed (Fig. In the P212121 form the was of the in the the form one in the and one dimer that formed a by a CEAS of three and as in the structure of (Fig. P. F. S. M. B. Zhang D. F. J. Mol. Biol. 1996; PubMed Scopus Google Scholar). The structure and of CEAS is to that of (Fig. and is in a of over of CEAS The is the most to its in over of CEAS in the is the over of CEAS The most the is the in CEAS of the of The of the is of at each of the (Fig. one CEAS the of a over and that the of the at the CEAS both one and an The was to be the most of P. F. S. M. B. Zhang D. F. J. Mol. Biol. 1996; PubMed Scopus Google Scholar), which may be to the tetrameric that with that enzyme The of CEAS which the of the that the has a arrangement around the active site to that of with the of of structural of the of CEAS and yeast pyruvate decarboxylase CEAS has sequence identity to the of the three of each is which is of the of such an arrangement to form the tightly for of the active of a in the crystal form is in with gel filtration which CEAS to in two solution The calculated native molecular of and to tetrameric and of CEAS N-terminal methionine the conditions of these in (Fig. The of two tightly over which are more to form a The has a size of on the and on the and to form the the each tightly dimer a of each a of with the of the dimer and with the the a of dimer is by a of By there are at the there are a considerable of in the the dimers, at the of the these may of the of the is best as a dimer of It is known for CEAS in the on substrate as in yeast an substrate an of the with to each D. S. G. S. Eur. J. Biochem. 2000; PubMed Scopus Google Scholar). This is with to the and in a of one of the associated as in the The of the CEAS is most to that of from Zymomonas mobilis D. S. G. G. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), which in such a manner and in which the is By comparison to yeast the of the are with to each the of the In to yeast tetrameric arrangement is more and for has been as the on comparison that CEAS tetrameric will be to ThP2 binding is two of the associated as with related enzymes such as The of one and the of the to with two ThP2 binding ThP2 binding is by a combination of and of a with a Mg2+ The Mg2+ is by the side of and the carbonyl of two diphosphate of ThP2 and a to the carbonyl of (Fig. This is to the binding identified as a conserved sequence in ThP2 enzyme A. Lett. PubMed Scopus Google Scholar). This sequence is in the and to the binding of the diphosphate of the ThP2. of the diphosphate directly to the of and from the The aminopyrimidine of the ThP2 is associated with of both the and the of the in the It is via and the carbonyl of and and the side chain of of the of the The ThP2 cofactor is observed in a “V” conformation (Fig. around the side chain of on ThP2-dependent enzymes that a is in a of ThP2 into high potential a protein (23Jordan F. Nat. Prod. Rep. 2003; 20: 184-201Crossref PubMed Scopus (201) Google Scholar). The of out is to an and from on yeast that may have an important effect on mechanism Zhang A. F. Biochemistry. 1998; 37: PubMed Scopus Google Scholar). and the a sulfate anion be identified in the density at a of from the nucleophilic carbon of ThP2. It is that sulfate the binding of the phosphate of The sulfate is at one by the side of and at by and at a by from the in the dimer (Fig. The sulfate is directly to the protein and is at a of from the nucleophilic of ThP2. This is for a these two as be from the reaction formed by ThP2 and (Fig. biosynthetic mechanism of that the at which l-arginine and the of proton and are A phosphate binding site has been identified in the crystal structure of complexed with U. G. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). The phosphate is in a of two side a and a The phosphate is from the of ThP2 in at a of which with the size of the substrate over the of The structure of yeast synthase a phosphate to the of ThP2 J. Mol. Biol. 2002; PubMed Scopus Google Scholar). the phosphate and the as a binding site for the group of the pyruvate substrate. The active site the proposed binding is containing the side of and The in are and It is in the presence of the side chain of into a of in the the CEAS active site is its of to By of the active site with side proposed in a role in are in CEAS with amino by and by It is difficult to mechanistic functions to in the CEAS active site and that CEAS on ThP2 for of the of the active site a of that may from the and the the proposed phosphate binding site, and may in of the of Khaleeli (12Khaleeli N. Li R. Townsend C.A. J. Am. Chem. Soc. 1999; 121: 9223-9224Crossref Scopus (82) Google Scholar) proposed an mechanism for the condensation of with l-arginine, initial nucleophilic attack of the ThP2 on the of and loss of the at the that addition of to with of at D.B. Townsend C.A. J. Chem. Soc. Chem. Commun. 1993; Scopus Google Scholar, A. Townsend C.A. J. Am. Chem. Soc. 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S. R. S. G. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar). the of the to for such the in from to for a more the and amino of the two reaction to in the of most ThP2-dependent either the is as in or is such as in reacts with substrate This in mechanism is on the presence of a proton to the In the has been shown to via a M. J. Am. Chem. Soc. 1996; Scopus Google Scholar). In benzoylformate has been shown to the with the of McLeish M.J. F. Kenyon G.L. M.S. Biochemistry. 2003; PubMed Scopus Google Scholar). to be such proton present in CEAS an an to of It that with CEAS the carbanion subsequently reacts to the group to give an The three binding the phosphate are to and the of phosphate This give an α,β-unsaturated acyl-ThP2 which as a for binding for the nucleophilic amino group in a for such an hydrolysis of the acyl-ThP2 the β-amino acid of the including proton are that the aminopyrimidine ring of ThP2 a key role in of the including of both and and addition of of of the and the of and of the for the at the Protein for with high