ABSTRACT
During the course of our research on the lead optimisation of the NBTI (Novel Bacterial Type II Topoisomerase Inhibitors) class of antibacterials, we discovered a series of tricyclic compounds that showed good Gram-positive and Gram-negative potency. Herein we will discuss the various subunits that were investigated in this series and report advanced studies on compound 1 (GSK945237) which demonstrates good PK and in vivo efficacy properties.
Subject(s)
Anti-Bacterial Agents/pharmacology , Heterocyclic Compounds, 3-Ring/pharmacology , Heterocyclic Compounds, 4 or More Rings/chemistry , Heterocyclic Compounds, 4 or More Rings/pharmacology , Topoisomerase II Inhibitors/chemistry , Topoisomerase II Inhibitors/pharmacology , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacokinetics , Chemistry Techniques, Synthetic , DNA Topoisomerases, Type II/chemistry , DNA Topoisomerases, Type II/metabolism , Dogs , Drug Evaluation, Preclinical/methods , ERG1 Potassium Channel/metabolism , Gram-Negative Anaerobic Bacteria/drug effects , Gram-Positive Bacteria/drug effects , Heterocyclic Compounds, 3-Ring/chemical synthesis , Heterocyclic Compounds, 3-Ring/chemistry , Pneumococcal Infections/drug therapy , Rats , Respiratory Tract Infections/drug therapy , Respiratory Tract Infections/microbiology , Topoisomerase II Inhibitors/pharmacokineticsABSTRACT
Despite the success of genomics in identifying new essential bacterial genes, there is a lack of sustainable leads in antibacterial drug discovery to address increasing multidrug resistance. Type IIA topoisomerases cleave and religate DNA to regulate DNA topology and are a major class of antibacterial and anticancer drug targets, yet there is no well developed structural basis for understanding drug action. Here we report the 2.1 A crystal structure of a potent, new class, broad-spectrum antibacterial agent in complex with Staphylococcus aureus DNA gyrase and DNA, showing a new mode of inhibition that circumvents fluoroquinolone resistance in this clinically important drug target. The inhibitor 'bridges' the DNA and a transient non-catalytic pocket on the two-fold axis at the GyrA dimer interface, and is close to the active sites and fluoroquinolone binding sites. In the inhibitor complex the active site seems poised to cleave the DNA, with a single metal ion observed between the TOPRIM (topoisomerase/primase) domain and the scissile phosphate. This work provides new insights into the mechanism of topoisomerase action and a platform for structure-based drug design of a new class of antibacterial agents against a clinically proven, but conformationally flexible, enzyme class.
Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , DNA Gyrase/chemistry , Quinolines/chemistry , Quinolines/pharmacology , Staphylococcus aureus/enzymology , Topoisomerase II Inhibitors , Anti-Bacterial Agents/metabolism , Apoenzymes/chemistry , Apoenzymes/metabolism , Arginine/metabolism , Aspartic Acid/metabolism , Binding Sites , Catalytic Domain , Ciprofloxacin/chemistry , Ciprofloxacin/metabolism , Crystallography, X-Ray , DNA/chemistry , DNA/metabolism , DNA Cleavage , DNA Gyrase/metabolism , DNA, Superhelical/chemistry , DNA, Superhelical/metabolism , Drug Design , Drug Resistance , Escherichia coli/enzymology , Manganese/metabolism , Models, Molecular , Protein Conformation , Quinolines/metabolism , Quinolones/chemistry , Quinolones/metabolism , Structure-Activity RelationshipABSTRACT
During the course of our research to find novel mode of action antibacterials, we discovered a series of hydroxyl tricyclic compounds that showed good potency against Gram-positive and Gram-negative pathogens. These compounds inhibit bacterial type IIA topoisomerases. Herein we will discuss structure-activity relationships in this series and report advanced studies on compound 1 (GSK966587) which demonstrates good PK and in vivo efficacy properties. X-ray crystallographic studies were used to provide insight into the structural basis for the difference in antibacterial potency between enantiomers.
Subject(s)
Bacteria/enzymology , Naphthyridines/chemistry , Naphthyridines/pharmacology , Topoisomerase II Inhibitors/chemistry , Topoisomerase II Inhibitors/pharmacology , Animals , Crystallography, X-Ray , Dogs , Enzyme Activation/drug effects , Haplorhini , Humans , Microbial Sensitivity Tests , Molecular Structure , RatsABSTRACT
As part of our wider efforts to exploit novel mode of action antibacterials, we have discovered a series of cyclohexyl-amide compounds that has good Gram positive and Gram negative potency. The mechanism of action is via inhibition of bacterial topoisomerases II and IV. We have investigated various subunits in this series and report advanced studies on compound 7 which demonstrates good PK and in vivo efficacy properties.
Subject(s)
Amides/chemistry , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , DNA Topoisomerases, Type II/chemistry , Gram-Negative Bacteria/drug effects , Gram-Positive Bacteria/drug effects , Topoisomerase II Inhibitors/chemistry , Amides/chemical synthesis , Amides/pharmacokinetics , Animals , Anti-Bacterial Agents/chemical synthesis , Binding Sites , Computer Simulation , DNA Topoisomerases, Type II/metabolism , Dogs , Haplorhini , Humans , Microbial Sensitivity Tests , Protein Structure, Tertiary , Rats , Structure-Activity Relationship , Topoisomerase II Inhibitors/chemical synthesis , Topoisomerase II Inhibitors/pharmacokineticsABSTRACT
We have identified a series of amino-piperidine antibacterials with a good broad spectrum potency. We report the investigation of various subunits in this series and advanced studies on compound 8. Compound 8 possesses good pharmacokinetics, broad spectrum antibacterial activity and demonstrates oral efficacy in a rat lung infection model.
Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , DNA Topoisomerases, Type II/chemistry , Dioxanes/chemistry , Dioxanes/pharmacology , Naphthyridines/chemistry , Naphthyridines/pharmacology , Piperidines/chemistry , Topoisomerase II Inhibitors/chemistry , Animals , Anti-Bacterial Agents/therapeutic use , DNA Topoisomerases, Type II/metabolism , Dioxanes/therapeutic use , Disease Models, Animal , Dogs , Haplorhini , Humans , Lung Diseases/drug therapy , Microbial Sensitivity Tests , Naphthyridines/therapeutic use , Piperidines/pharmacology , Piperidines/therapeutic use , Rats , Structure-Activity Relationship , Topoisomerase II Inhibitors/pharmacology , Topoisomerase II Inhibitors/therapeutic useABSTRACT
Previously it has been postulated that the pyochelin-Fe outer membrane transporter, FptA, is involved in the uptake of catechol-substituted cephalosporins in Pseudomonas aeruginosa. Iron uptake and antibacterial activity studies on different mutants showed clearly that FptA is unable to bind and transport these antibiotics.
Subject(s)
Bacterial Outer Membrane Proteins/metabolism , Catechols/chemistry , Cephalosporins/pharmacology , Pseudomonas aeruginosa/drug effects , Receptors, Cell Surface/metabolism , Cephalosporins/chemistryABSTRACT
The sequencing of the first complete bacterial genome in 1995 heralded a new era of hope for antibacterial drug discoverers, who now had the tools to search entire genomes for new antibacterial targets. Several companies, including GlaxoSmithKline, moved back into the antibacterials area and embraced a genomics-derived, target-based approach to screen for new classes of drugs with novel modes of action. Here, we share our experience of evaluating more than 300 genes and 70 high-throughput screening campaigns over a period of 7 years, and look at what we learned and how that has influenced GlaxoSmithKline's antibacterials strategy going forward.
Subject(s)
Anti-Bacterial Agents/therapeutic use , Bacteria/drug effects , Bacterial Infections/drug therapy , Animals , Bacteria/genetics , Bacterial Infections/microbiology , Drug Design , Genomics , HumansABSTRACT
Retapamulin MICs of > or =2 microg/ml were noted for 6 of 5,676 S. aureus recent clinical isolates evaluated. The ABC proteins VgaAv and VgaA were found to be responsible for the reduced susceptibility to pleuromutilins exhibited by these six isolates.
Subject(s)
ATP-Binding Cassette Transporters/genetics , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Drug Resistance, Bacterial , Staphylococcus aureus/drug effects , Diterpenes/pharmacology , Drug Resistance, Bacterial/genetics , Humans , Microbial Sensitivity Tests , Molecular Sequence Data , Polycyclic Compounds , Sequence Analysis, DNA , Staphylococcus aureus/genetics , Staphylococcus aureus/isolation & purification , PleuromutilinsABSTRACT
Fluoroquinolone drugs such as moxifloxacin kill bacteria by stabilizing the normally transient double-stranded DNA breaks created by bacterial type IIA topoisomerases. Previous crystal structures of Staphylococcus aureus DNA gyrase with asymmetric DNAs have had static disorder (with the DNA duplex observed in two orientations related by the pseudo-twofold axis of the complex). Here, 20-base-pair DNA homoduplexes were used to obtain crystals of covalent DNA-cleavage complexes of S. aureus DNA gyrase. Crystals with QPT-1, moxifloxacin or etoposide diffracted to between 2.45 and 3.15â Å resolution. A G/T mismatch introduced at the ends of the DNA duplexes facilitated the crystallization of slightly asymmetric complexes of the inherently flexible DNA-cleavage complexes.
Subject(s)
DNA Cleavage , DNA Gyrase/chemistry , Etoposide/chemistry , Fluoroquinolones/chemistry , Heterocyclic Compounds, 4 or More Rings/chemistry , Spiro Compounds/chemistry , Staphylococcus aureus/enzymology , Base Sequence , Crystallization , Crystallography, X-Ray , Molecular Sequence Data , MoxifloxacinABSTRACT
New antibacterials are needed to tackle antibiotic-resistant bacteria. Type IIA topoisomerases (topo2As), the targets of fluoroquinolones, regulate DNA topology by creating transient double-strand DNA breaks. Here we report the first co-crystal structures of the antibacterial QPT-1 and the anticancer drug etoposide with Staphylococcus aureus DNA gyrase, showing binding at the same sites in the cleaved DNA as the fluoroquinolone moxifloxacin. Unlike moxifloxacin, QPT-1 and etoposide interact with conserved GyrB TOPRIM residues rationalizing why QPT-1 can overcome fluoroquinolone resistance. Our data show etoposide's antibacterial activity is due to DNA gyrase inhibition and suggests other anticancer agents act similarly. Analysis of multiple DNA gyrase co-crystal structures, including asymmetric cleavage complexes, led to a 'pair of swing-doors' hypothesis in which the movement of one DNA segment regulates cleavage and religation of the second DNA duplex. This mechanism can explain QPT-1's bacterial specificity. Structure-based strategies for developing topo2A antibacterials are suggested.
Subject(s)
Anti-Bacterial Agents/chemistry , Antineoplastic Agents/chemistry , DNA Gyrase/chemistry , Etoposide/chemistry , Fluoroquinolones/chemistry , Staphylococcus aureus/enzymology , Topoisomerase II Inhibitors/chemistry , Anti-Bacterial Agents/pharmacology , Antineoplastic Agents/pharmacology , DNA Gyrase/genetics , DNA Gyrase/metabolism , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , Drug Resistance, Bacterial , Etoposide/pharmacology , Fluoroquinolones/pharmacology , Models, Molecular , Molecular Structure , Moxifloxacin , Staphylococcus aureus/chemistry , Staphylococcus aureus/drug effects , Topoisomerase II Inhibitors/pharmacologyABSTRACT
This chapter describes two key strategies for the discovery of new antibacterial agents and illustrates the critical role played by genomics in each. The first approach is genomic target-based screening. Comparative genomics and bioinformatics are used to identify novel, selective antibacterial targets of the appropriate antibacterial spectrum. Genetic technologies integral for the success of this approach, such as essentiality testing, are also described. An unprecedented number of novel targets have been discovered via this approach, and a plethora of examples are discussed. This section concludes with the case history of a target successfully progressed from identification by genomics, to high-throughput screening, and onto proof of concept in curing experimental infections. The second approach is based on screening for compounds with antibacterial activity and then employing a broad variety of newer technologies to identify the molecular target of the antibacterial agent. The advantage of this approach is that compounds already possess antibacterial activity, which is a property often challenging to engineer into molecules obtained from enzyme-based screening approaches. The recent development of novel biochemical and genomic technologies that facilitate identification and characterization of the mode of action of these agents has made this approach as attractive as the genomic target-based screening strategy.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacteriological Techniques/methods , Genome, Bacterial , Anti-Bacterial Agents/isolation & purification , Bacteria/drug effects , Bacteria/genetics , Bacteria/pathogenicity , Base Composition , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Genomics , Humans , Microbial Sensitivity TestsABSTRACT
The discovery of novel antibiotic classes has not kept pace with the growing threat of bacterial resistance. Antibiotic candidates that act at new targets or via distinct mechanisms have the greatest potential to overcome resistance; however, novel approaches are also associated with higher attrition and longer timelines. This uncertainty has contributed to the withdrawal from antibiotic programs by many pharmaceutical companies. Genomic approaches have not yielded satisfactory results, in part due to nascent knowledge about unprecedented molecular targets, the challenge of achieving antibacterial activity by lead optimization of enzyme inhibitors, and the limitations of compound screening libraries for antibacterial discovery. Enhanced diversity of compound screening banks, entry into new chemical space, and new screening technologies are currently being exploited to improve hit rates for antibacterial discovery. Antibacterial compound lead optimization faces hurdles associated with the high plasma exposures required for efficacy. Lead optimization would be enhanced by the identification of new antibiotic classes with improved tractability and by expanding the predictability of in vitro safety assays. Implementing multiple screening and target identification strategies is recommended for improving the likelihood of discovering new antibacterial compounds that address unmet needs.
Subject(s)
Anti-Bacterial Agents/therapeutic use , Bacterial Infections/drug therapy , Drug Discovery/methods , Drug Resistance, Bacterial/drug effects , Animals , Drug Design , Drug Evaluation, Preclinical/methods , Drug Industry/methods , Humans , Microbial Sensitivity TestsABSTRACT
We developed a homogenous microtiter based assay using the cationic dye 3, 3'-Diethyloxacarbocyanine iodide, DiOC2(3), to measure the effect of compounds on membrane potential in Staphylococcus aureus. In a screen of 372 compounds from a synthetic compound collection with anti-Escherichia coli activity due to unknown modes of action at least 17% demonstrated potent membrane activity, enabling rapid discrimination of nuisance compounds.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacteriological Techniques/methods , High-Throughput Screening Assays/methods , Membrane Potentials/drug effects , Staphylococcus aureus/drug effects , Carbocyanines/metabolism , Escherichia coli/drug effects , Inhibitory Concentration 50ABSTRACT
Quinolone antibacterials have been used to treat bacterial infections for over 40 years. A crystal structure of moxifloxacin in complex with Acinetobacter baumannii topoisomerase IV now shows the wedge-shaped quinolone stacking between base pairs at the DNA cleavage site and binding conserved residues in the DNA cleavage domain through chelation of a noncatalytic magnesium ion. This provides a molecular basis for the quinolone inhibition mechanism, resistance mutations and invariant quinolone antibacterial structural features.
Subject(s)
Acinetobacter baumannii/enzymology , DNA Topoisomerase IV/chemistry , Enzyme Inhibitors/chemistry , Quinolones/chemistry , DNA Topoisomerase IV/pharmacology , Enzyme Inhibitors/pharmacology , Models, Molecular , Protein Structure, Quaternary , Protein Structure, Tertiary , Quinolones/pharmacologyABSTRACT
Fluoroquinolones are an important class of antibiotics for the treatment of infections arising from the gram-positive respiratory pathogen Streptococcus pneumoniae. Although there is evidence supporting interspecific lateral DNA transfer of fluoroquinolone target loci, no studies have specifically been designed to assess the role of intraspecific lateral transfer of these genes in the spread of fluoroquinolone resistance. This study involves a comparative evolutionary perspective, in which the evolutionary history of a diverse set of S. pneumoniae clinical isolates is reconstructed from an expanded multilocus sequence typing data set, with putative recombinants excluded. This control history is then assessed against networks of each of the four fluoroquinolone target loci from the same isolates. The results indicate that although the majority of fluoroquinolone target loci from this set of 60 isolates are consistent with a clonal dissemination hypothesis, 3 to 10% of the sequences are consistent with an intraspecific lateral transfer hypothesis. Also evident were examples of interspecific transfer, with two isolates possessing a parE-parC gene region arising from viridans group streptococci. The Spain 23F-1 clone is the most dominant fluoroquinolone-nonsusceptible clone in this set of isolates, and the analysis suggests that its members act as frequent donors of fluoroquinolone-nonsusceptible loci. Although the majority of fluoroquinolone target gene sequences in this set of isolates can be explained on the basis of clonal dissemination, a significant number are more parsimoniously explained by intraspecific lateral DNA transfer, and in situations of high S. pneumoniae population density, such events could be an important means of resistance spread.
Subject(s)
DNA Topoisomerases, Type II/genetics , Drug Resistance, Bacterial/genetics , Evolution, Molecular , Gene Transfer, Horizontal , Streptococcus pneumoniae/genetics , Amino Acid Sequence , Anti-Infective Agents/pharmacology , Base Sequence , DNA Gyrase/genetics , Fluoroquinolones/pharmacology , Molecular Sequence Data , Phylogeny , Sequence Homology, Amino Acid , Streptococcus pneumoniae/drug effects , Streptococcus pneumoniae/enzymology , Streptococcus pneumoniae/isolation & purificationABSTRACT
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.
Subject(s)
Hydroxymethylglutaryl-CoA Synthase/chemistry , Staphylococcus aureus/enzymology , Amino Acid Motifs , Amino Acid Sequence , Asparagine/chemistry , Binding Sites , Catalysis , Cholesterol/metabolism , Cloning, Molecular , Crystallography, X-Ray , Cysteine/chemistry , Dimerization , Escherichia coli/metabolism , Histidine/chemistry , Hydroxymethylglutaryl-CoA Synthase/genetics , Kinetics , Models, Chemical , Models, Molecular , Molecular Sequence Data , Protein Conformation , Sequence Homology, Amino AcidABSTRACT
Type II topoisomerases bind to DNA at the catalytic domain across the DNA gate. DNA gyrases also bind to DNA at the non-homologous C-terminal domain of the GyrA subunit, which causes the wrapping of DNA about itself. This unique mode of DNA binding allows gyrases to introduce the negative supercoils into DNA molecules. We have investigated the biochemical characteristics of Staphylococcus aureus (S. aureus) gyrase. S. aureus gyrase is known to require high concentrations of potassium glutamate (K-Glu) for its supercoiling activity. However, high concentrations of K-Glu are not required for its relaxation and decatenation activities. This is due to the requirement of high concentrations of K-Glu for S. aureus gyrase-mediated wrapping of DNA. These results suggest that S. aureus gyrase can bind to DNA at the catalytic domain independent of K-Glu concentration, but high concentrations of K-Glu are required for the binding of the C-terminal domain of GyrA to DNA and the wrapping of DNA. Thus, salt modulates the DNA binding mode and the catalytic activity of S. aureus gyrase. Quinolone drugs can stimulate the formation of covalent S. aureus gyrase-DNA complexes, but high concentrations of K-Glu inhibit the formation of S. aureus gyrase-quinolone-DNA ternary complexes. In the absence of K-Glu, ternary complexes formed with S. aureus gyrase cannot arrest replication fork progression in vitro, demonstrating that the formation of a wrapped ternary complex is required for replication fork arrest by a S. aureus gyrase-quinolone-DNA ternary complex.
Subject(s)
DNA Gyrase/metabolism , DNA Replication , DNA, Bacterial/metabolism , Glutamates/pharmacology , Quinolones/metabolism , Staphylococcus aureus/metabolism , Catalysis , Protein Binding , Staphylococcus aureus/drug effects , Staphylococcus aureus/enzymology , Staphylococcus aureus/geneticsABSTRACT
Antibiotic efflux is an important mechanism of resistance in pathogenic bacteria. Here we describe the identification and characterization of a novel chromosomally encoded multidrug resistance efflux protein in Staphylococcus aureus, MdeA (multidrug efflux A). MdeA was identified from screening an S. aureus open reading frame expression library for resistance to antibiotic compounds. When overexpressed, MdeA confers resistance on S. aureus to a range of quaternary ammonium compounds and antibiotics, but not fluoroquinolones. MdeA is a 52-kDa protein with 14 predicted transmembrane segments. It belongs to the major facilitator superfamily and is most closely related, among known efflux proteins, to LmrB of Bacillus subtilis and EmrB of Escherichia coli. Overexpression of mdeA in S. aureus reduced ethidium bromide uptake and enhanced its efflux, which could be inhibited by reserpine and abolished by an uncoupler. The mdeA promoter was identified by primer extension. Spontaneous mutants selected for increased resistance to an MdeA substrate had undergone mutations in the promoter for mdeA, and their mdeA transcription levels were increased by as much as 15-fold. The mdeA gene was present in the genomes of all six strains of S. aureus examined. Uncharacterized homologs of MdeA were present elsewhere in the S. aureus genome, but their overexpression did not mediate resistance to the antibacterials tested. However, MdeA homologs were identified in other bacteria, including Bacillus anthracis, some of which were shown to be functional orthologs of MdeA.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B/genetics , Bacterial Proteins/genetics , Chromosomes, Bacterial/genetics , Staphylococcus aureus/genetics , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA Primers , Molecular Sequence Data , Mutation , Phylogeny , Plasmids/genetics , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Many bacteria employ the nonmevalonate pathway for synthesis of isopentenyl diphosphate, the monomer unit for isoprenoid biosynthesis. However, gram-positive cocci exclusively use the mevalonate pathway, which is essential for their growth (E. I. Wilding et al., J. Bacteriol. 182:4319-4327, 2000). Enzymes of the mevalonate pathway are thus potential targets for drug intervention. Uniquely, the enterococci possess a single open reading frame, mvaE, that appears to encode two enzymes of the mevalonate pathway, acetoacetyl-coenzyme A thiolase and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. Western blotting revealed that the mvaE gene product is a single polypeptide in Enterococcus faecalis, Enterococcus faecium, and Enterococcus hirae. The mvaE gene was cloned from E. faecalis and was expressed with an N-terminal His tag in Escherichia coli. The gene product was then purified by nickel affinity chromatography. As predicted, the 86.5-kDa mvaE gene product catalyzed both the acetoacetyl-CoA thiolase and HMG-CoA reductase reactions. Temperature optima, DeltaH(a) and K(m) values, and pH optima were determined for both activities. Kinetic studies of acetoacetyl-CoA thiolase implicated a ping-pong mechanism. CoA acted as an inhibitor competitive with acetyl-CoA. A millimolar K(i) for a statin drug confirmed that E. faecalis HMG-CoA reductase is a class II enzyme. The oxidoreductant was NADP(H). A role for an active-site histidine during the first redox step of the HMG-CoA, reductase reaction was suggested by the ability of diethylpyrocarbonate to block formation of mevalonate from HMG-CoA, but not from mevaldehyde. Sequence comparisons with other HMG-CoA reductases suggest that the essential active-site histidine is His756. The mvaE gene product represents the first example of an HMG-CoA reductase fused to another enzyme.