Pseudomonas Mevalonii Research Articles

Development and application of computational methods for the study of protein dynamics with Pm-HMGR as a model system.

Continuous atomistic descriptions of enzyme mechanisms and atomistic dynamics are crucial to the characterization of protein function and dysfunction—a common cause of disease—but unattainable with current techniques, necessitating novel methods which leverage computational-experimental synergy. Computational studies can provide a wealth of information, but carry little weight without experimental validation. Thus, coupling time-resolved crystallographic studies of enzyme mechanisms with computational mechanistic investigation through molecular dynamics (MD) simulation and predictive model generation provides the opportunity to combine approaches. We demonstrate and refine a novel Markov State-informed Multilinear Singular Value Decomposition (MSiMSVD) method with the Pseudomonas mevalonii HMG CoA Reductase (PmHMGR) pathway as a model system. MSiMSVD couples time-resolved crystallographic studies of enzyme mechanisms with computational mechanistic investigation through MD simulation and predictive model generation. Application of the MSiMSVD method to slow dynamical events-such as the PmHMGR 2nd hydride transfer-is limited by the ability of force fields to reproduce atomistic behavior at a transition state. This need can be met via the generation of a Transition State Force Field (TSFF) with a program such as Quantum-guided Molecular Mechanics (Q2MM). However, a frequently encountered problem in the optimization of TSFFs for biomolecules is the high dimensionality of the optimization surface producing local rather than global minima. To address this, we implement a constrained particle swarm algorithm hybridized with a basic genetic algorithm to automate the optimization of TSFFs with Q2MM. The Q2MM and the MSiMSVD methods will become accessible to non-experts for a wide range of systems, such as large biomolecules. Thus far, initial Markov State Models generated from MD simulation of the PmHMGR 2nd hydride transfer indicate that more sampling is necessary to cover the transition state conformation, i.e. the structure of the activated complex.

Automated fitting of transition state force fields for biomolecular simulations.

The generation of surrogate potential energy functions (PEF) that are orders of magnitude faster to compute but as accurate as the underlying training data from high-level electronic structure methods is one of the most promising applications of fitting procedures in chemistry. In previous work, we have shown that transition state force fields (TSFFs), fitted to the functional form of MM3* force fields using the quantum guided molecular mechanics (Q2MM) method, provide an accurate description of transition states that can be used for stereoselectivity predictions of small molecule reactions. Here, we demonstrate the applicability of the method for fit TSFFs to the well-established Amber force field, which could be used for molecular dynamics studies of enzyme reaction. As a case study, the fitting of a TSFF to the second hydride transfer in Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl coenzyme A reductase (PmHMGR) is used. The differences and similarities to fitting of small molecule TSFFs are discussed.

Utilizing a pH-dependent reaction triggering method to elucidate the mechanism of bacterial HMG-CoA reductase using time-resolved crystallography

HMG-CoA reductase in Pseudomonas mevalonii (PmHMGR) utilizes the co-substrates mevalonate and coenzyme-A (CoA) and 2 NAD+ cofactors to generate HMG-CoA. Having shown to be an essential enzyme for the survival of gram-positive antibiotic resistant pathogenic bacteria, studying its mechanism in detail will be beneficial in developing novel antibacterials. Details about this mechanism such as the conformational changes that allow intermediate formation, the cofactor exchange at the active site and product release remain unknown.

Computational Study of PmHMGR Thiohemiacetal Breakdown Transition State

3-hydroxyl-3-methylglutaryl CoA reductase (HMGR) catalyzes the rate determining step in the isoprenoid pathway using two NADH molecules. Even though this enzyme is targeted by statins, the atomistic details of the mechanism are still unknown. This objective can be met by combination of computational biophysics and experimental validations through time-resolved Laue crystallography for all steps of the reaction. The catalysis of HMGR can be broken down into three major chemical transformations including two hydride transfers and a thiohemiacetal breakdown step. Moreover, since two cofactor molecules are required, a cofactor exchange process also occurs after the first hydride transfer and before the second hydride transfer. Thiohemiacetal is a known intermediate resulting from the first hydride transfer step. However, the product of the thiohemiacetal breakdown step hypothesized to be mevaldehyde, has not been experimentally validated. This theoretical study explores the atomic details of the enzyme and ligands for the thiohemiacetal breakdown step using the Pseudomonas mevalonii HMGR. Theozymes and QM/MM ONIOM models were used to identify the transition state were performed to reveal the catalytic residues. These models have revealed that the Nδ of His381 is involved in stabilizing the negative charge in the thiolate anion. Ser85 forms a hydrogen bonding network with the first amide of thiohemiacetal ligand, a function performed by His381 in the preceding step first hydride transfer. Transition state force fields (TSFF) using the quantum guided molecular mechanics (Q2MM) program will then be used to perform µsec timescale molecular dynamics simulations. This project focuses on not only developing computational biophysics methods for studying enzyme mechanisms but also uses time-resolved x-ray crystallography to obtain snapshots along the HMGR reaction pathway which involves multiple transformations of the ligand in the same active site, a large flap domain movement, and a cofactor exchange.

Microsecond timescale MD simulations at the transition state of PmHMGR predict remote allosteric residues.

Understanding the mechanisms of enzymatic catalysis requires a detailed understanding of the complex interplay of structure and dynamics of large systems that is a challenge for both experimental and computational approaches. More importantly, the computational demands of QM/MM simulations mean that the dynamics of the reaction can only be considered on a timescale of nanoseconds even though the conformational changes needed to reach the catalytically active state happen on a much slower timescale. Here we demonstrate an alternative approach that uses transition state force fields (TSFFs) derived by the quantum-guided molecular mechanics (Q2MM) method that provides a consistent treatment of the entire system at the classical molecular mechanics level and allows simulations at the microsecond timescale. Application of this approach to the second hydride transfer transition state of HMG-CoA reductase from Pseudomonas mevalonii (PmHMGR) identified three remote residues, R396, E399 and L407, (15–27 Å away from the active site) that have a remote dynamic effect on enzyme activity. The predictions were subsequently validated experimentally via site-directed mutagenesis. These results show that microsecond timescale MD simulations of transition states are possible and can predict rather than just rationalize remote allosteric residues.

A novel role for coenzyme A during hydride transfer in 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

In this study, we take advantage of the ability of HMG-CoA reductase (HMGR) from Pseudomonas mevalonii to remain active while in its crystallized form to study the changing interactions between the ligands and protein as the first reaction intermediate is created. HMG-CoA reductase catalyzes one of the few double oxidation-reduction reactions in intermediary metabolism that take place in a single active site. Our laboratory has undertaken an exploration of this reaction space using structures of HMG-CoA reductase complexed with various substrate, nucleotide, product, and inhibitor combinations. With a focus in this publication on the first hydride transfer, our structures follow this reduction reaction as the enzyme converts the HMG-CoA thioester from a flat sp(2)-like geometry to a pyramidal thiohemiacetal configuration consistent with a transition to an sp(3) orbital. This change in the geometry propagates through the coenzyme A (CoA) ligand whose first amide bond is rotated 180° where it anchors a web of hydrogen bonds that weave together the nucleotide, the reaction intermediate, the enzyme, and the catalytic residues. This creates a stable intermediate structure prepared for nucleotide exchange and the second reduction reaction within the HMG-CoA reductase active site. Identification of this reaction intermediate provides a template for the development of an inhibitor that would act as an antibiotic effective against the HMG-CoA reductase of methicillin-resistant Staphylococcus aureus.

Characterization of an HMG-CoA Reductase from Listeria monocytogenes That Exhibits Dual Coenzyme Specificity

HMG-CoA reductase (HMGR) is an enzyme critical for cellular cholesterol synthesis in mammals and isoprenoid synthesis in certain eubacteria, catalyzing the NAD(P)H-dependent reduction of HMG-CoA to mevalonate. We have isolated the gene encoding HMG-CoA reductase from Listeria monocytogenes and expressed the recombinant 6x-His-tagged form in Escherichia coli. Using NAD(P)(H), the enzyme catalyzes HMG-CoA reduction approximately 200-fold more efficiently than mevalonate oxidation in vitro. The purified enzyme exhibits dual coenzyme specificity, utilizing both NAD(H) and NADP(H) in catalysis; however, catalytic efficiency using NADP(H) is approximately 200 times greater than when using NAD(H). The statins mevinolin and mevastatin are weak inhibitors of L. monocytogenes HMG-CoA reductase, requiring micromolar concentrations for inhibition. Three-dimensional modeling reveals that the overall structure of L. monocytogenes HMG-CoA reductase is likely similar to the known structure of the class II enzyme from Pseudomonas mevalonii. It appears that the enzyme has catalytic amino acids in analogous positions that likely play similar roles and also has a flap domain that brings a catalytic histidine into the active site. However, in L. monocytogenes HMG-CoA reductase histidine 143 and methionine 186 are present in the putative NAD(P)(H)-selective site, possibly interacting with the 2' phosphate of NADP(H) or 2' hydroxyl of NAD(H) and providing the active site architecture necessary for dual coenzyme specificity.

The 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductases

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase catalyzes the conversion of HMG-CoA to mevalonate and is a target for anti-hypercholesterolemic drugs (statins).

Identification of novel eubacteria from spent mushroom compost (SMC) waste by DNA sequence typing: ecological considerations of disposal on agricultural land

A small study was undertaken to examine the microbiological characteristics of spent mushroom compost (SMC), which is the major waste by-product of the mushroom industry and which is regularly disposed off by application to agricultural land. The primary aim of this study was to examine SMC for the presence of faecal bacterial pathogens, including Campylobacter spp., Salmonella spp. and Listeria monocytogenes. Secondly it was desirable to quantify bacterial and fungal populations within SMC, and also qualitatively identify the diversity of bacterial populations within SMC, through employment of rDNA PCR and direct sequencing techniques on the culturable microflora. Conventional microbiological analyses of SMC material ( n=30) from six commercial operations in both Northern Ireland and the Republic of Ireland, failed to detect Salmonella spp , Listeria spp. or Campylobacter spp. in any of the SMC material examined. Total aerobic plate counts gave a mean count of log 10 7.01 colony forming units (cfu) per gram SMC material (range: log 10 6.53–7.52 cfu/g). Fungal counts gave a mean count of log 10 4.57 cfu per gram SMC material (range: log 10 3.93–4.98 cfu/g). From a total of greater than 50 colony picks, a total of 12 bacterial morphotypes were identified and were further examined by employment of partial 16S rRNA gene amplification and sequencing techniques, yielding several genera and species, including Bacillus licheniformis, Bacillus subtilis, Klebsiella/Enterobacter sp. Microbacterium sp. Paenibacillus lentimorbus, Pseudomonas mevalonii, Sphingobacterium multivorum and Stenotrophomonas sp. This is the first preliminary report on the microbial diversity of SMC waste and demonstrates the presence of several species that have not been previously described in SMC, in addition to two potentially novel species within the genera Microbacterium and Stenotrophomonas. It is thereby important to examine the ecological microbe–microbe and plant–microbe interactions that are occurring between the native bacterial soil flora and those added annually (theoretically estimated at approximately 10 18 cells) through the application of SMC. Such studies would be beneficial in helping to ascertain the ecological consequences involved in the disposal of SMC waste on agricultural land.

Crystal Structure of a Statin Bound to a Class II Hydroxymethylglutaryl-CoA Reductase

Hydroxymethylglutaryl-CoA (HMG-CoA) reductase is the primary target in the current clinical treatment of hypercholesterolemias with specific inhibitors of the "statin" family. Statins are excellent inhibitors of the class I (human) enzyme but relatively poor inhibitors of the class II enzymes of important bacterial pathogens. To investigate the molecular basis for this difference we determined the x-ray structure of the class II Pseudomonas mevalonii HMG-CoA reductase in complex with the statin drug lovastatin. The structure shows lovastatin bound in the active site and its interactions with residues critically involved in catalysis and substrate binding. Binding of lovastatin also displaces the flap domain of the enzyme, which contains the catalytic residue His-381. Comparison with the structures of statins bound to the human enzyme revealed a similar mode of binding but marked differences in specific interactions that account for the observed differences in affinity. We suggest that these differences might be exploited to develop selective class II inhibitors for use as antibacterial agents against pathogenic microorganisms.

Pseudomonas mevalonii 3-hydroxy-3methylglutaryl-CoA lyase

Pseudomonas mevalonii 3-hydroxy-3methylglutaryl-CoA lyase

Investigation of the conserved lysines of Syrian hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase.

Sequence analysis has revealed two classes of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Crystal structures of ternary complexes of the Class II enzyme from Pseudomonas mevalonii revealed lysine 267 critically positioned at the active site. This observation suggested a revised catalytic mechanism in which lysine 267 facilitates hydride transfer from reduced coenzyme by polarizing the carbonyl group of HMG-CoA and subsequently of bound mevaldehyde, an inference supported by mutagenesis of lysine 267 to aminoethylcysteine. For this mechanism to be general, Class I HMG-CoA reductases ought also to possess an active site lysine. Three lysines are conserved among all Class I HMG-CoA reductases. The three conserved lysines of Syrian hamster HMG-CoA reductase were mutated to alanine. All three mutant enzymes had reduced but detectable activity. Of the three conserved lysines, sequence alignments implicate lysine 734 of the hamster enzyme as the most likely cognate of P. mevalonii lysine 267. Low activity of enzyme K734A did not reflect an altered structure. Substrate recognition was essentially normal, and both circular dichroism spectroscopy and analytical ultracentrifugation implied a native structure. Enzyme K734A also formed an active heterodimer when coexpressed with inactive mutant enzyme D766N. We infer that a lysine is indeed essential for catalysis by the Class I HMG-CoA reductases and that the revised mechanism for catalysis is general for all HMG-CoA reductases.

Aminoethylcysteine can replace the function of the essential active site lysine of Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl coenzyme A reductase.

The biodegradative 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase of Pseudomonas mevalonii catalyzes the NAD(+)-dependent conversion of (S)-HMG-CoA to (R)-mevalonate. Crystallographic analysis of abortive ternary complexes of this enzyme revealed lysine 267 located at a position in the active site, suggesting that it might serve as the general acid/base for catalysis. Site-directed mutagenesis and subsequent chemical derivatization were therefore employed to investigate this active site lysine. Replacement of lysine 267 by alanine, histidine, or arginine resulted in mutant enzymes that lacked detectable activity. Lysine 267 was next replaced by the lysine analogues aminoethylcysteine and carboxyamidomethylcysteine. Using instead of the wild-type enzyme the fully active, cysteine-free mutant enzyme C156A/C296A, lysine 267 was first replaced by cysteine. Cysteine 267 of mutant enzyme C156A/C296A/K267C was then treated with bromoethylamine or iodoacetamide to insert aminoethylcysteine (AEC) or carboxyamidomethylcysteine at position 267. The carboxyamidomethylcysteine derivative was inactive, whereas mutant enzyme C156A/C296A/K267AEC exhibited high catalytic activity. That aminoethylcysteine, but not other basic amino acids, can replace the function of lysine 267 documents both the importance of this residue and the requirement for a precisely positioned positive charge at the active site of the enzyme.

Purification and characterization of the heteromeric transcriptional activator MvaT of the Pseudomonas mevalonii mvaAB operon.

The mvaAB operon of Pseudomonas mevalonii encodes HMG-CoA reductase (EC 1.1.1.88) and HMG-CoA lyase (EC 4.1.3.4), enzymes that catalyze the initial reactions of mevalonate catabolism in this organism. Expression of this operon is regulated by the constitutively expressed transcriptional activator protein MvaT that binds in vitro to an upstream regulatory element. Mevalonate is essential for activation of transcription in vivo, and in vitro data demonstrated that MvaT binds to the mvaAB cis-regulatory element in the absence of mevalonate with a Kd,app of 2 nM. Purification of MvaT enriched for two polypeptides of approximate molecular mass 15 kDa and 16 kDa, designated P15 and P16. MvaT, assayed by its DNA-binding activity, comigrated with P15 and P16 during DNA-affinity chromatography, size-exclusion chromatography, and sucrose density gradient centrifugation. P15 and P16 also comigrated during denaturing isoelectric focusing of purified MvaT. Treatment of MvaT with dimethylsuberimidate formed a 31-kDa polypeptide complex that contained N-terminal sequences from P15 and P16. The apparent association of P15 and P16 in solution and their copurification with MvaT activity strongly suggests that MvaT is comprised of these two subunits. Size-exclusion chromatography gave an estimated molecular mass for MvaT of 33 kDa. A partial DNA sequence of the P16 gene was obtained using PCR employing degenerate primers directed against the N-termini of P15 and P16. P16 appears to be comprised of at least 128 aminoacyl residues having a predicted molecular mass of 14.3 kDa.

3-hydroxy-3-methylglutaryl coenzyme A reductase of Sulfolobus solfataricus: DNA sequence, phylogeny, expression in Escherichia coli of the hmgA gene, and purification and kinetic characterization of the gene product.

The gene (hmgA) for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) from the thermophilic archaeon Sulfolobus solfataricus P2 was cloned and sequenced. S. solfataricus HMG-CoA reductase exhibited a high degree of sequence identity (47%) to the HMG-CoA reductase of the halophilic archaeon Haloferax volcanii. Phylogenetic analyses of HMG-CoA reductase protein sequences suggested that the two archaeal genes are distant homologs of eukaryotic genes. The only known bacterial HMG-CoA reductase, a strictly biodegradative enzyme from Pseudomonas mevalonii, is highly diverged from archaeal and eukaryotic HMG-CoA reductases. The S. solfataricus hmgA gene encodes a true biosynthetic HMG-CoA reductase. Expression of hmgA in Escherichia coli generated a protein that both converted HMG-CoA to mevalonate and cross-reacted with antibodies raised against rat liver HMG-CoA reductase. S. solfataricus HMG-CoA reductase was purified in 40% yield to a specific activity of 17.5 microU per mg at 50 degrees C by a sequence of steps that included heat treatment, ion-exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography. The final product was homogeneous, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The substrate was (S)- not (R)-HMG-CoA; the reductant was NADPH not NADH. The Km values for HMG-CoA (17 microM) and NADPH (23 microM) were similar in magnitude to those of other biosynthetic HMG-CoA reductases. Unlike other HMG-CoA reductases, the enzyme was stable at 90 degrees C and was optimally active at pH 5.5 and 85 degrees C.

A 3-hydroxy-3-methylglutaryl-CoA lyase gene in the photosynthetic bacterium Rhodospirillum rubrum

A 1.2 kb long DNA segment from Rhodospirillum rubrum has been sequenced (EMBL/GenBank accession number: U41280). This DNA segment includes the first sequenced gene for a putative 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase, termed hmgL, from a photosynthetic organism. The sequenced segment also contains a ribosome-binding site and two clusters of possible −35 and −10 promotor sequences preceding the hmgL gene. Translation of the gene would yield a 303 amino-acid-long protein with a calculated molecular weight of 31.1 kDa. This protein shows 55–60% identity and approx. 75% similarity, including conservative substitutions, with the three eukaryotic and the single prokaryotic HMG-CoA lyases which previously have been sequenced. The R. rubrum enzyme showed stronger homology to the chicken HMG-CoA lyase than to the other bacterial protein. Significant sequence similarity was also found with homocitrate synthases from nitrogen-fixing prokaryotes. In contrast to the other sequenced prokaryotic HMG-CoA lyase (from Pseudomonas mevalonii), the R. rubrum hmgL does not seem to appear in an operon together with a HMG-CoA reductase. The hmgL gene was transcribed in photosynthetically grown cells as judged by amplification of cDNAs synthesised from DNA-free total RNA. In addition, HMG-CoA lyase activity was found in R. rubrum cells grown anaerobically in the light with leucine as the carbon source.

3-Hydroxy-3-methylglutaryl-coenzyme A reductase from Haloferax volcanii: purification, characterization, and expression in Escherichia coli

Prior work from this laboratory characterized eukaryotic (hamster) and eubacterial (Pseudomonas mevalonii) 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductases. We report here the characterization of an HMG-CoA reductase from the third domain, the archaea. HMG-CoA reductase of the halobacterium Haloferax volcanii was initially partially purified from extracts of H. volcanii. Subsequently, a portion of the H. volcanii lovastatin (formerly called mevinolin) resistance marker mev was subcloned into the Escherichia coli expression vector pT7-7. While no HMG-CoA reductase activity was detectable following expression in E. coli, activity could be recovered after extracts were exposed to 3 M KCl. Following purification to electrophoretic homogeneity, the specific activity of the expressed enzyme, 24 microU/mg, equaled that of homogeneous hamster or P. mevalonii HMG-CoA reductase. Activity was optimal at pH 7.3. Kms were 66 microM (NADPH) and 60 microM [(S)-HMG-CoA]. (R)-HMG-CoA and lovastatin inhibited competitively with (S)-HMG-CoA. H. volcanii HMG-CoA reductase also catalyzed the reduction of mevaldehyde [optimal activity at pH 6.0; Vmax 11 microU/mg; Kms 32 microM (NADPH), 550 microM [(R,S)-mevaldehyde]] and the oxidative acylation of mevaldehyde [optimal activity at pH 8.0; Vmax 2.1 microU/mg; Kms 350 microM (NADP+), 300 microM (CoA), 470 microM [(R,S)-mevaldehyde]]. These properties are comparable to those of hamster and P. mevalonii HMG-CoA reductases, suggesting a similar catalytic mechanism.

Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-CoA lyase: Testing the function of the active site cysteine by site-directed mutagenesis

Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-CoA lyase is affinity labeled by 2-butynoyl-CoA; peptide sequence analysis demonstrates C237 to be the site of modification [Hruz et al. (1992) Biochemistry 31, 6842-6847]. In order to evaluate whether C237 functions in the chemistry of hydroxymethylglutaryl-CoA cleavage, cassette mutagenesis has been employed to alter wild-type DNA to encode serine or alanine at residue 237. ESR measurements indicate that the purified mutant enzymes bind stoichiometric amounts of the spin-labeled substrate analog, R.CoA, which has been established as a competitive inhibitor. Binding affinities measured with C237S (Kd = 92 microM) and C237A (Kd = 97 microM) lyases are comparable to that observed with wild-type lyase. The rotational dynamics of R.CoA bound to mutant enzymes are also very similar to those for R.CoA bound to wild-type lyase. These observations suggest that the mutant enzymes are structurally intact. In view of this demonstrated structural integrity, it is significant that the VmaxS of C237A and C237S are approximately 4 x 10(4)- and approximately 725-fold lower, respectively, than the value measured for wild-type hydroxymethylglutaryl-CoA lyase. The C237S enzyme exhibits a Km = 53 microM for substrate; this value is only 2-fold higher than the Km of the wild-type enzyme. Additionally, we report that the residual activity in C237S hydroxymethylglutaryl-CoA lyase is unaffected by 2-butynoyl-CoA under conditions which support inactivation of wild-type enzyme. These results are consistent with an active site assignment to C237, confirming the prediction based on the affinity labeling/peptide mapping data.(ABSTRACT TRUNCATED AT 250 WORDS)

Crystal structure of Pseudomonas mevalonii HMG-CoA reductase at 3.0 angstrom resolution.

The rate-limiting step in cholesterol biosynthesis in mammals is catalyzed by 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, a four-electron oxidoreductase that converts HMG-CoA to mevalonate. The crystal structure of HMG-CoA reductase from Pseudomonas mevalonii was determined at 3.0 angstrom resolution by multiple isomorphous replacement. The structure reveals a tightly bound dimer that brings together at the subunit interface the conserved residues implicated in substrate binding and catalysis. These dimers are packed about a threefold crystallographic axis, forming a hexamer with 23 point group symmetry. Difference Fourier studies reveal the binding sites for the substrates HMG-CoA and reduced or oxidized nicotinamide adenine dinucleotide [NAD(H)] and demonstrate that the active sites are at the dimer interfaces. The HMG-CoA is bound by a domain with an unusual fold, consisting of a central alpha helix surrounded by a triangular set of walls of beta sheets and alpha helices. The NAD(H) is bound by a domain characterized by an antiparallel beta structure that defines a class of dinucleotide-binding domains.

Crystallization of HMG-CoA reductase from Pseudomonas mevalonii.

Crystals of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase from Pseudomonas mevalonii have been grown by vapor diffusion in hanging drops at pH 6.7 using ammonium sulfate as the precipitant. Serial dilution seeding and manipulation of glycerol concentration were both used to obtain crystals larger than 1.0 mm. The crystals are cubic, space group I4(1)32, with a = 229.4 A. A V(m) value of 2.71 A(3) Da(-l) indicates 96 molecules per unit cell with two molecules in the asymmetric unit. These crystals diffract to 2.8 A with conventional X-ray sources, and beyond 2.4 A with synchrotron radiation.