Staphylococcus aureus 3-hydroxy-3-methylglutaryl-CoA synthase: crystal structure and mechanism.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, a member of the family of acyl-condensing enzymes, catalyzes the first committed step in the mevalonate pathway and is a potential target for novel antibiotics and cholesterol-lowering agents. The Staphylococcus aureus mvaS gene product (43.2 kDa) was overexpressed in Escherichia coli, purified to homogeneity, and shown biochemically to be an HMG-CoA synthase. The crystal structure of the full-length enzyme was determined at 2.0-A resolution, representing the first structure of an HMG-CoA synthase from any organism. HMG-CoA synthase forms a homodimer. The monomer, however, contains an important core structure of two similar alpha/beta motifs, a fold that is completely conserved among acyl-condensing enzymes. This common fold provides a scaffold for a catalytic triad made up of Cys, His, and Asn required by these enzymes. In addition, a crystal structure of HMG-CoA synthase with acetoacetyl-CoA was determined at 2.5-A resolution. Together, these structures provide the structural basis for an understanding of the mechanism of HMG-CoA synthase.

Two pathways have been identified for the synthesis of isopentenyl pyrophosphate (IPP), 1 the mevalonate pathway ( Fig.  1) and the glyceraldehyde 3-phosphate-pyruvate pathway (1,2). IPP is the central precursor of isoprenoids, which are ubiquitous in nature and comprise a family of over 23,000 compounds. The principal end product of IPP metabolism in higher animals is cholesterol, and in bacteria, end products include the lipid carrier undecaprenol, involved in cell wall biosynthesis (3), menaquinones and ubiquinones, involved in electron transport (4,5), and carotenoids (6). Mammals and archaea possess only the mevalonate pathway for IPP biosynthesis, whereas both pathways occur amongst plants. Among pathogenic bacteria, the low GϩC Gram-positive cocci possess genes that encode for enzymes of the mevalonate pathway and are essential for the growth of Staphylococcus aureus and Streptococcus pneumoniae (7,8). The best characterized enzyme of the mevalonate pathway in both eukaryotes and prokaryotes is 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, and crystal structures have been solved for both human (10) and bacterial (11) HMG-CoA reductase. The eukaryotic HMG-CoA reductase is the target of the statin class of cholesterol-lowering agents (12). Prokaryotic HMG-CoA reductases are over 4 orders of magnitude less sensitive to inhibition by these drugs (13). Thus the pathway represents a potential target for antibacterial agents against life-threatening, multidrug-resistant Gram-positive cocci.
HMG-CoA synthase (EC 4.1.3.5) is the enzyme preceding HMG-CoA reductase in the mevalonate pathway (Fig. 1). HMG-CoA synthase catalyzes the biological Claisen condensation of acetyl-CoA with acetoacetyl-CoA and is a member of a superfamily of acyl-condensing enzymes that includes ␤-ketothiolases, fatty acid synthases (␤-ketoacyl-acyl carrier protein synthase), and polyketide synthases. All members of the superfamily use a cysteine as a nucleophile and shuttle reaction intermediates through CoA molecules (14 -16). Despite low sequence homology, the overall structures of these condensing enzymes are conserved, displaying a common fold with a highly structured ␣/␤ motif and extended loop regions. However, significant differences in the structure of the active sites are apparent, reflecting substrate and mechanistic differences and suggesting functional divergence.
The amino acid sequences of the staphylococcal and streptococcal HMG-CoA synthases are well conserved (44% identity), although the Gram-positive enzymes exhibit little identity (20%) with those of human and avian HMG-CoA synthases. Despite the low overall identity, the amino acid residues involved in the acetylation and condensation reactions are conserved among bacterial and eukaryotic HMG-CoA synthases. In common with other members of this superfamily, kinetic, protein chemistry, and spectroscopic studies of native and sitedirected mutants of the mammalian HMG-CoA synthase suggested the involvement of a cysteine residue in the acyl transfer from the carrier CoA to the carbonyl acceptor (16 -18). The role of the cysteine residue is well established on the basis of mechanistic evidence, although no direct structural data from an HMG-CoA synthase from any organism have been reported.
In this report, we show that the mvaS gene product from S. aureus (43.2 kDa) is an HMG-CoA synthase and report the three-dimensional crystal structure of the full-length homodimeric enzyme complexed with acetoacetyl-CoA. The structure provides the molecular basis for a potential reaction mechanism and provides insight to aid rational drug design.

EXPERIMENTAL PROCEDURES
Cloning and Overexpression of S. aureus HMG-CoA Synthase-The HMG-CoA synthase gene (mvaS) was amplified from S. aureus WCUH29 genomic DNA (NCIMB 40771) using Pwo polymerase (Roche Applied Science) and ligated into pET28a(ϩ) (Novagen) that had been digested with NcoI and EcoRI and dephosphorylated with alkaline phosphatase. The insert was sequenced to confirm that no errors were introduced during amplification, and the recombinant plasmid, pET-MvaS, was transformed into Escherichia coli BL21(DE3).
Protein Purification-E. coli harboring pET-MvaS were grown to mid-log phase and induced with isopropyl-1-thio-␤-D-galactopyranoside. Cells were lysed, and insoluble material was pelleted by centrifugation. Ammonium sulfate was added to the soluble fraction to 70% saturation, and insoluble material was removed by centrifugation. The supernatant was concentrated and applied to a Mono Q column, and bound protein was eluted with a 0 -1 M NaCl linear gradient. Fractions containing HMG-CoA synthase activity (0.30 -0.35 M NaCl) were pooled, determined to be Ͼ95% pure by SDS-PAGE, and stored in 20 mM triethanolamine-HCl, pH 7.5, 5 mM dithiothreitol, 1 mM EDTA, 50% glycerol at Ϫ20°C. Protein concentrations were determined by the Bradford dye binding assay (Bio-Rad) using bovine serum albumin as a standard. N-terminal sequencing was performed at the GlaxoSmith-Kline Protein Core Facility.
Steady-state Kinetics-HMG-CoA synthase activity was assayed spectrophotometrically by monitoring the acetyl-CoA-dependent rate of acetoacetyl-CoA depletion at 300 nm. An extinction coefficient of 7,200 M Ϫ1 cm Ϫ1 was calculated by an end point assay, which reflects the amount of acetoacetyl-CoA in the enolate form in the presence of 20 mM MgCl 2 . This compares with a value of 13,600 M Ϫ1 cm Ϫ1 calculated previously (19). Reactions were performed in 100 mM HEPES, pH 8.0, containing 0.1 mM EDTA using a Spectramax Plus (Molecular Devices) spectrophotometer. Kinetic data were fit to the appropriate rate equations by using the nonlinear regression function of SigmaPlot (Jandel Scientific). Individual saturation curves were fit to Equation 1 to determine the kinetic parameters (V and V/K) for the substrates. At the higher concentration range of ammonium sulfate, the crystals grew to 0.3 ϫ 0.3 ϫ 0.1 mm and were space group P2 1 . The hexagonal crystals were more stable, diffracted to 2-Å resolution, and were used for initial structure determination. The P2 1 crystal form diffracted to 2.5-Å resolution and were sensitive to handling. However, it was found that only the monoclinic crystals contained acetoacetyl-CoA in the structure. For data collection, crystals were soaked in 2.4 M ammonium sulfate, 0.1 M Tris-HCl, pH 8.0, and 25% (v/v) glycerol for a few seconds and then frozen at Ϫ160°C. Data sets were collected on a Rigaku RU200 rotating anode with an Raxis IV x-ray detector or were collected at the Advanced Photon Source, beamline 17-ID (Industrial Macromolecular Crystallography Association Collaborative Access Team). All data were processed with HKL2000 package (20) and described in Table  I (see below).
Phase Determination-Heavy atom derivatives were prepared by soaking the hexagonal crystals in reservoir solution containing 1 mM K 2 PtCl 4 for 24 h. These crystals did not show any platinum binding, but the space group and unit cell were altered. Therefore the K 2 PtCl 4 crystals were treated as the initial native data set for phasing. A second crystal was soaked in 1 mM K 2 PtCl 4 for 24 h, and then 1 mM UO 2 Ac 2 was added to the reservoir for soaking overnight. The uranium data were collected on a rotating anode and at the APS to take advantage of  the different x-ray absorption properties of uranium with respect to x-ray energy. Initial heavy atom positions were found by inspection, confirmed by using SOLVE (21), and refined using MLPHARE (22). Phase calculation, solvent flattening, and phase extension to 2.0 Å were done with DM (23).
Model Building and Refinement-The solvent-flattened electron density map was used to build a model in the program O (24). The Apo model was refined with the program CNX (25) using the initial native data set. The monoclinic form of HMG-CoA synthase containing the cofactor was solved via molecular replacement with the program AMORE (26) using the refined Apo structure as a search model. Difference Fourier maps clearly show the acetoacetyl-CoA binding at the active site. Further refinement was completed with REFMAC5 (27). The figures were made with PhotoShop (Adobe), ISIS/Draw (MDL Information Systems, Inc), Molscript (28), Bobscript (29), Raster3D (30), and Pymol (31).
Coordinates-The coordinates have been deposited with the Protein Data Bank (accession codes for apoHMG-CoA synthase and acetoacetyl-CoA-HMG-CoA synthase are 1TVZ and 1TXT, respectively).

RESULTS AND DISCUSSION
Cloning and Isolation of S. aureus HMG-CoA Synthase-Induction of the mvaS gene of S. aureus cloned into pET28a(ϩ) resulted in the expression of the gene product to ϳ30% of total soluble protein. Typical yields were 7.5 mg of over 95% homogenous protein/liter of induced culture. HMG-CoA synthase purifies as a homodimer with molecular weight of 86000 Daltons (388 amino acid residues/monomer). Liquid chromatography mass spectrometry analysis indicated that the purified protein had a molecular weight of 43,077, which was within the limits of error to the expected mass of 43,073 for the full-length protein minus the N-terminal methionine residue. Certain preparations of the protein had a mass of 32 Da greater than expected, consistent with structural observations showing that the active site cysteine residue contained two additional oxygen atoms and with the observation that the protein over time lost catalytic activity.
Steady-state Kinetics-S. aureus HMG-CoA synthase exhibits activity exclusively with acetyl-CoA as the acyl group donor and acetoacetyl-CoA as the substrates. The Michaelis-Menten constants determined for acetyl-CoA and acetoacetyl-CoA have values of 350 Ϯ 50 and 3 Ϯ 1 M with a k cat of 0.50 Ϯ 0.02 s Ϫ1 . Additional experiments show that acyl-CoA substrates larger than two carbon units do not enzymatically condense with acetoacetyl-CoA.
Three-dimensional Structure Determination and Description-The purified enzyme crystallized in two space groups (P6 5 22 or P2 1 ). The P6 5 22 crystal form contained two molecules in the asymmetric unit. Upon soaking these crystals in K 2 PtCl 4 to find derivatives, the c axis was halved, and the space group was changed to P6 4 22 with one molecule in the asymmetric unit. The P2 1 crystal form contained four molecules in the asymmetric unit and was more sensitive to handling. Since no platinum heavy atom sites were found in the P6 4 22 crystals, the data had been collected to 2.0-Å resolution, and the uranium data set was isomorphous, these crystals were treated as native during the structure solution with the result that only one polypeptide needed to be built and refined. The 2.0-Å resolution apoenzyme model of HMG-CoA synthase was determined in space group P6 4 22 using heavy atom isomorphous replacement anomalous scattering and solvent flattening (Table I). The hexagonal model contained one complete polypeptide chain with the noncrystallographic 2-fold parallel to the crystallographic c axis and 198 water molecules. The P2 1 structure was determined by molecular replacement and includes two complete homodimers, four acetoacetyl-CoA molecules, and 388 water molecules. Only the P2 1 crystal form contained acetoacetyl-CoA in the active site. The overall structure of the P6 4 22 form was identical to that of the P2 1 form with the exception of the substrate in the active site.
HMG-CoA synthase forms a homodimer of two 43.2-kDa polypeptides (388 residues/monomer) related by a 2-fold axis and forms a rectangular box shape with the approximate dimensions 56 ϫ 63 ϫ 78 Å (Fig. 2). Electron density for acetoacetyl-CoA was visible within each monomer and defined the active site. Each HMG-CoA synthase monomer consists of two structural regions referred to as the upper and lower regions. The upper region is built around a five-layered core structure, ␣-␤-␣-␤-␣, where each ␣ comprises two ␣-helices and each ␤ is a mixed ␤-sheet. This core structure contains an obvious internal duplication of two similar ␣/␤ motifs. ␤1, ␤4, ␤5, ␤6, ␤7, ␣3, ␣5, and ␣6 define one half, and ␤9, ␤12, ␤13, ␤14, ␣7, ␣8, and ␣10 define the second half of the core. These two parts can be optimally superimposed by considering 77 equivalent residues to give a root mean square deviation of 2.2 Å on C ␣ -atoms. The lower region is defined by a three-stranded ␤-sheet (␤2, ␤3, ␤15), two two-stranded ␤-sheets (␤10 and ␤11 and ␤16 and ␤17, respectively), and three helices, ␣1, ␣2, and ␣12. The lower region does not contain any substructure or pseudo-symmetry. However, the interface of the upper and lower regions defines the acetoacetyl-CoA-binding site.
The monomers form an extensive interface, as indicated by the large surface area of ϳ2580 Å 2 that is buried upon formation of the dimer. Several extensive monomer-monomer interactions can be defined. A major interaction is through strand ␤5, which interacts with ␤5 of the other monomer to form a 10-stranded ␤-sheet in the dimer. Arg-103 of strand ␤5 extends across the dimer interface to form a salt bridge to Glu-312 of strand ␤14. The C-terminal portion of ␣6 interacts with its symmetry mate to further define the dimer interface by a salt bridge between Asp-123 and Arg-128.
Other Condensing Enzymes-The pseudo-symmetric motif of the core structure within the upper region was originally observed in thiolase (9). This core structure is also seen in chalcone synthase (32) and fatty acid ␤-ketoacyl-ACP synthases I, II, and III (KAS I, II, and III) (33-35). (Fig. 3). The upper region of HMG-CoA synthase and KAS III (E. coli FabH) can be superimposed with a root mean square deviation of 1.8 Å for 265 equivalent C ␣ atoms, implying a common evolutionary origin. A structure-based sequence alignment shows that the catalytic triad Cys, His, and Asn is part of the conserved core structure and provides for the first step common to the respec-tive reactions. The respective lower regions show the highest differences among the condensing enzymes. These differences primarily result from large amino acid insertions at various points in the respective proteins. HMG-CoA synthase has 67 amino acids at the C terminus; chalcone synthase has a 36amino-acid insert near the N terminus, and thiolase has a 100-amino-acid insert near the middle. No large inserts and having the smallest lower region structure distinguish FabH.
Active Site and Mechanism-The interface of the upper and lower regions defines an active site tunnel, which is long (ϳ13 Å) and narrow, suitable for substrate binding. The tunnel is the binding site for acetyl-CoA, the first substrate. After CoA release, acetoacetyl-CoA, the second substrate, occupies the tunnel. The well defined electron density of acetoacetyl-CoA occupying the tunnel allows the characterization of important residues for substrate binding and catalysis (Fig. 2). At the tunnel entrance, Lys-32, Lys-202, Lys-242, and Lys-238 form interactions with the phosphates of acetoacetyl-CoA. Asp-29 within helix ␣2 forms a hydrogen bond to the amine group of the adenosine moiety. One side of the adenosine packs against the face of helix ␣2; the opposite side of adenosine is solventexposed. Van der Waals contacts dominate the majority of interactions between the pantothenate unit of CoA and the  (32), and 1PXT (9), respectively). b, structure-based sequence alignment. Conserved cysteine, histidine, and asparagine residues are red. The core structure is cyan and green; cyan indicates helices, and green indicates ␤-strands.
Overall, the mechanism of HMG-CoA synthase consists of three steps occurring via a ping-pong mechanism: first, the acylation of a cysteine by acetyl-CoA; second, the Claison con-densation of acetyl-Cys with acetoacetyl-CoA; and finally, the release of HMG-CoA from the enzyme. The upper region acts as a scaffold holding the key residues in position for the first step of the reaction, which is common to all condensing enzymes: the acetylation of a Cys residue. In HMG-CoA synthase, Cys-111 sits at the bottom of the tunnel and provides the essential thiol group in the enzymatic reaction for HMG-CoA synthase. The HMG-CoA Synthase, Crystal Structure 44887 electron density shows oxidation of the sulfhydryl of Cys-111 to sulfinic acid (-SO2H) during the crystallization. However, this oxidatation does allow for the trapping of acetoacetyl-CoA in the active site. Cys-111, His-233, and Asn-275 form a triad similar to that found in FabH and chalcone synthase (Fig. 3).
His-233 and Asn-275 flank Cys-111. In the thiolase structure, the Asn and His are swapped spatially (Figs. 3b and 4). His-233 is positioned to act as a base activating Cys-111 that can then initiate the acetyl transfer by a nucleophilic attack at the carbonyl carbon of acetyl-CoA. As in the other condensing enzymes, the acetylation occurs via a tetrahedral intermediate and is stabilized by residues in the active site. A functional survey of invariant acidic residues identified Asp-83, Asp-139, and Asp-184 as important in acetyl-S-enzyme formation, which involves the formation and directed collapse of a tetrahedral adduct (36). The crystal structure shows that these residues are directly or indirectly a part of the active site. Only Asp-184 is involved in stabilizing the adduct; the other two aspartates are important to maintaining the tertiary structure of the protein. Asp-184 interacts with Ser-307 through hydrogen-bonding interactions to the hydroxyl group of Ser-307 and the amide proton of Gly-308. The backbone amide proton of Gly-308 and the hydroxyl group of Ser-307 define an oxyanion-stabilizing hole for the carbonyl oxygen of acetyl-CoA during the tetrahedral intermediate. Ser-307 is part of a ␤-turn located between strands, ␤13 and ␤14, that contains a cis peptide between Gly-308 and Ser-309. Asp-184 is also important in stabilizing the cis peptide. Asp-139 and Asp-83 are buried and form hydrogen bonds to the amide backbone hydrogen of residues Glu-79 and Arg-187, respectively, providing important interactions to maintain the structural integrity of the enzyme and not a part of the oxyanion hole.
The subsequent enzymatic step diverges significantly in the different acyl-condensing enzymes. In HMG-CoA synthase, the acetylated cysteine acts as a nucleophile and attacks the second substrate. This Claisen condensation is significantly different from that seen in KAS enzymes, in which the second substrate acts as a nucleophile by releasing CO 2 and attacks the acetylated cysteine residues of the KAS enzymes. In addition to being involved in the acylation of Cys-111, the invariant histidine (S. aureus His-233) appears to be important in binding of acetoacetyl CoA. As seen in the crystal structure, the penultimate carbonyl of acetoacetyl-CoA is within hydrogen-bonding distance to His-233 (Fig. 4). The end carbonyl of acetoacetyl-CoA is difficult to place due to poor electron density. The invariant Tyr-143 would suggest that it is in a position to hydrogen-bond to the end carbonyl of acetoacetyl-CoA, holding the second substrate in place. However, mutagenesis of the corresponding tyrosine in the avian enzyme (Y163L) did not show a significant decrease in the catalytic activity (37). Also, the product stereochemistry would require the end carbonyl to be pointing away from Tyr-143. The invariant Glu-79 has been shown in the avian enzyme (Glu-95) to act as a general acid/ base in the condensation reaction. Glu-79 is in an ideal position to act as a base and activate the acyl group of Cys-111 for a nucleophilic attack upon acetoacetyl-CoA, creating the HMG-CoA enzyme complex. The presence of structured water around the active site suggests that the release of HMG-CoA from the enzyme occurs via hydrolysis by the ordered water molecules.
Based on our structure, additional mutagenesis and biochemical experiments can be proposed to test these models and further probe the mechanism. In addition, the structure of HMG-CoA synthase will facilitate the structure-based design of alternative drugs to cholesterol-lowering therapies or to novel antibiotics to the Gram-positive cocci. Crystallization efforts on human HMG-CoA synthase have been unsuccessful, whereas the S. aureus HMG-CoA synthase readily crystallizes and will prove useful for drug development against a different enzyme in the mevalonate pathway.