Biomedical subjects

V W Rodwell

Publications and source records attributed to V W Rodwell.

At least 37 records · Page 2Linked to original sources

PubMed1995

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.

Journal Article↗

PubMed1994

Phosphorylation of Ser871 impairs the function of His865 of Syrian hamster 3-hydroxy-3-methylglutaryl-CoA reductase.

The attenuation of catalytic activity that accompanies phosphorylation of Ser871 of Syrian hamster 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) reflects primarily the introduction of negative charge (Omkumar, R. V., Darnay, B. G., and Rodwell, V. W. (1994) J. Biol. Chem. 269, 6810-6814). To investigate how a negative charge at position 871 attenuates activity, we phosphorylated wild-type and mutant HMG-CoA reductases and assayed reduction of the putative intermediate mevaldehyde to mevalonate. We observed attenuated activity when the phosphorylated wild-type enzyme was assayed in the presence or absence of coenzyme A, but not when assayed in the presence of desthio-CoA. These observations recall the behavior of mutant enzyme H865Q, for which coenzyme A inhibits, whereas desthio-CoA stimulates mevaldehyde reduction (Frimpong, K. F., and Rodwell, V. W. (1994) J. Biol. Chem. 269, 11478-11483). Catalysis of mevaldehyde reduction by mutant enzyme H865Q was unaffected by phosphorylation. By contrast, mutant enzymes H860Q and H868Y, in which nearby, but noncatalytic, histidines had been mutated, exhibited wild-type behavior upon phosphorylation. We conclude that the introduction of negative charge at position 871 impairs the function of His865, presumably by a specific electrostatic interaction. We propose a novel mechanism by which phosphorylation regulates activity. Phosphorylation of the terminal serine of the consensus AGxLV(K/R)SHMxxNRS motif of eukaryotic HMG-CoA reductases attenuates activity by impairing the ability of the catalytic histidine to protonate the CoAS- anion formed during the reductive deacylation of HMG-CoA to mevaldehyde.

Amino Acid Sequence↗

PubMed1994

Catalysis by Syrian hamster 3-hydroxy-3-methylglutaryl-coenzyme A reductase. Proposed roles of histidine 865, glutamate 558, and aspartate 766.

We employed the overexpressed catalytic domains of wild-type Syrian hamster 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) and of mutant enzymes E558Q, H865Q, and D766N to investigate the roles of Glu558, His865, and Asp786 in catalysis. Five reactions were studied: reductive deacylation of HMG-CoA or of mevaldyl-CoA to mevalonate, reduction of mevaldehyde to mevalonate, oxidation of mevaldyl-CoA to HMG-CoA, and oxidative acylation of mevaldehyde to HMG-CoA. While only the wild-type enzyme catalyzed all five reactions, mutant enzymes E558Q and H865Q catalyzed other reactions at significant rates. Mutant enzyme D766N, although apparently structurally similar to the wild-type enzyme, was inactive for all five reactions. While an ineffective catalyst for the overall reaction, mutant enzyme H865Q catalyzed the reduction of mevaldehyde to mevalonate at about the wild-type rate. Coenzyme A, which stimulated mevaldehyde reduction by the wild-type enzyme, inhibited mutant enzyme H865Q, apparently due to an impaired ability to protonate the coenzyme A thioanion, CoAS-. Based on these data, we infer that Glu558, His865, and Asp766 function in catalysis at the indicated stage of the overall reaction, [formula: see text] and we propose a revised mechanism for catalysis by mammalian HMG-CoA reductases.

Amino Acid Sequence↗

PubMed1994

Modulation of Syrian hamster 3-hydroxy-3-methylglutaryl-CoA reductase activity by phosphorylation. Role of serine 871.

Attenuation of Syrian hamster 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) activity by in vitro phosphorylation was studied using AMP-activated protein kinase and wild-type and mutant forms of HMG-CoA reductase. The only residue of the wild-type enzyme phosphorylated was Ser871. Substrates protected against kinase-mediated attenuation of activity, consistent with substrate-induced conformational changes at the C-terminal region. Although close to the catalytic histidine His865, Ser871 appears to play no direct role in catalysis or substrate recognition. Mutant enzymes S871A, S871H, S871N, and S871Q exhibited from 62-106% of wild-type activity and had wild-type Km values for HMG-CoA and NADPH. Replacement of Ser871 by aspartate or glutamate, but not by glutamine, asparagine, histidine, or tyrosine, severely attenuated activity. Attenuation of catalytic activity that accompanies phosphorylation thus appears to result primarily from the introduction of negative charge, not merely steric hindrance. Other than the wild-type enzyme, only mutant enzyme S871T was phosphorylated, and phosphorylation was accompanied by attenuation of activity. The AMP-activated kinase thus can also phosphorylate threonyl residues.

AMP-Activated Protein Kinases↗

PubMed1994

The active site of hamster 3-hydroxy-3-methylglutaryl-CoA reductase resides at the subunit interface and incorporates catalytically essential acidic residues from separate polypeptides.

We employed site-directed mutagenesis based on sequence comparisons and characterization of purified mutant enzymes to identify Glu558 and Asp766 of Syrian hamster 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) as essential for catalysis. Mutant enzymes E558D, E558Q, and D766N had wild-type Km values for (S)-HMG-CoA and NADPH, but exhibited less than 0.5% of the wild-type catalytic activity. The inactive mutant polypeptides E558Q and D766N nevertheless can associate to generate an active enzyme. In vitro, 6% of the wild-type activity was observed when mutant polypeptides E558D and D766N were mixed in the absence of chaotropic agents. When mutant polypeptides E558Q and D766N were co-expressed in Escherichia coli, the resulting purified enzyme had 25% of wild-type activity. Hamster HMG-CoA reductase thus is a two-site, dimeric enzyme whose subunits associate to form an active site in which each monomer contributes at least one residue (e.g. Glu558 from one monomer and Asp766 from the other). The wild-type enzyme behaves as a dimer during size exclusion chromatography and has one HMG-CoA binding site per monomer. Syrian hamster HMG-CoA reductase thus appears to be a homodimer with two active sites which are located at the subunit interface.

Acyl Coenzyme A↗

PubMed1993

His865 is the catalytically important histidyl residue of Syrian hamster 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

Involvement in catalysis of a histidyl residue of Syrian hamster 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase was suggested by the ability of diethyl pyrocarbonate to abolish catalytic activity, accompanying spectral changes, and reactivation by hydroxylamine. The 7 histidines present in the catalytic domain of the hamster enzyme were changed to glutamine (His474, His487, His634, His751, His860, and His865), lysine (His865), or tyrosine (His868). Overexpression in Escherichia coli yielded six soluble mutant proteins, one insoluble protein (H634Q), and one which was degraded in vivo (H487Q). Following purification to homogeneity, mutant enzymes H474Q, H751Q, H860Q, and H868Y had essentially wild-type catalytic activity, while mutant enzymes H865K and H865Q had less than 0.6% wild-type activity. The low activity of mutant enzymes H865K and H865Q is unlikely to reflect altered structural integrity since both chromatographed on affinity supports like wild-type enzyme and had Km values for (S)-HMG-CoA (31 and 16 microM) and for NADPH (60 and 24 microM) close to those for wild-type enzyme (31 and 52 microM for (S)-HMG-CoA and NADPH, respectively). His865 of hamster HMG-CoA reductase, the histidine of the consensus Leu-Val-Xaa-Ser-His-Met-Xaa-Xaa-Asn-Arg-Ser motif and the only histidine conserved among the catalytic domains of all HMG-CoA reductases, thus appears to be a general acid/base functional in catalysis.

Amino Acid Sequence↗

PubMed1993

Syrian hamster 3-hydroxy-3-methylglutaryl-coenzyme A reductase expressed in Escherichia coli: production of homogeneous protein.

When overexpressed in Escherichia coli, the catalytic domain of Syrian hamster 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) is catalytically active, but exhibits major heterogeneity. This heterogeneity reflects deletion of about 60 aminoacyl residues from the C-terminus, presumably a result of proteolytic cleavage or premature termination of translation. With the intent of separating the intact and truncated proteins via immunoaffinity chromatography, we constructed the expression phagemid pKFT7-21. This construct encodes the catalytic domain of Syrian hamster HMG-CoA reductase with the C-terminal extension Glu-Glu-Phe, an epitope recognized by a specific antibody. Following overexpression, the modified catalytic domain RcatEEF had high catalytic activity and exhibited no heterogeneity. It therefore was possible to purify RcatEEF to over 95% homogeneity without resorting to immunoaffinity chromatography. The yield of homogeneous protein averaged 20-25 mg per liter of cells with a final specific activity of up to 40 mumol NADPH oxidized per minute per milligram. The EEF modification thus should prove useful for the purification of the catalytic domains of other eukaryotic HMG-CoA reductases which exhibit heterogeneity.

Amino Acid Sequence↗

PubMed1992

Identification of the catalytically important histidine of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

We identify His381 of Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase as the basic residue functional in catalysis. The catalytic domain of 20 HMG-CoA reductases contains a single conserved histidine (His381 of the P. mevalonii enzyme). Diethyl pyrocarbonate inactivated the P. mevalonii enzyme, and hydroxylamine partially restored activity. We changed His381 to alanine, lysine, asparagine, and glutamine. The mutant proteins were overexpressed, purified to homogeneity, and characterized. His381 mutant enzymes were not inactivated by diethyl pyrocarbonate. All four mutant enzymes exhibited wild-type crystal morphology and chromatographed on substrate affinity supports like wild-type enzyme. The mutant enzymes had low catalytic activity (Vmax 0.06-0.5% that of wild-type enzyme), but Km values approximated those for wild-type enzyme. For wild-type enzyme and mutant enzymes H381A, H381N, and H381Q, Km values at pH 8.1 were 0.45, 0.27, 3.7, and 0.71 mM [(R,S)-mevalonate]; 0.05, 0.03, 0.20, and 0.11 mM [coenzyme A]; 0.22, 0.14, 0.81, and 0.62 mM [NAD+]. Km values at pH 11 for wild-type enzyme and mutant enzyme H381K were 0.32 and 0.75 mM [(R,S)-mevalonate]; 0.24 and 0.50 mM [coenzyme A]; 0.15 and 1.23 mM [NAD+]. Both pK values for the enzyme-substrate complex increased relative to wild-type enzyme (by 1-2.5 pH units for pK1 and by 0.5-1.3 pH units for pK2). For mutant enzyme H381K, the pK1 of 10.2 is consistent with lysine acting as a general base at high pH. His381 of P. mevalonii HMG-CoA reductase, and consequently the histidine of the consensus Leu-Val-Lys-Ser-His-Met-Xaa-Xaa-Asn-Arg-Ser motif of the catalytic domain of eukaryotic HMG-CoA reductases, thus is the general base functional in catalysis.

Amino Acid Sequence↗

PubMed1992

Biosynthesis and characterization of (S)-and (R)-3-hydroxy-3-methylglutaryl coenzyme A.

(S)-3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA), the physiologic substrate of HMG-CoA reductase and of HMG-CoA lyase, is available commercially only as (R,S)-HMG-CoA, a mixture of diastereomers. To provide (S)-HMG-CoA for our continuing investigation of HMG-CoA reductase, we used homogeneous, overexpressed Pseudomonas mevalonii HMG-CoA reductase (EC 1.1.1.88) and an NAD(+)-regenerating system to convert (R)-mevalonate to (S)-HMG-CoA with an overall yield in excess of 50%. We also used P. mevalonii HMG-CoA lyase (EC 4.1.3.4) to prepare (R)-HMG-CoA from (R,S)-HMG-CoA. Each diastereomer was then isolated by ion-exchange chromatography. Large-scale preparations provide for economical production of (S)-HMG-CoA, particularly when recovered coenzyme A is recycled. (S)-HMG-CoA was evaluated as a substrate, and (R)-HMG-CoA as an inhibitor, for the P. mevalonii enzymes HMG-CoA reductase and HMG-CoA lyase, and for Syrian hamster HMG-CoA reductase (EC 1.1.1.34). For both HMG-CoA reductases, (R)-HMG-CoA inhibited competitively with respect to (S)-HMG-CoA. The ratio Ki/Km was 0.7 +/- 0.1 and 0.6 +/- 0.2 for the bacterial and hamster enzymes, respectively. By contrast, (R)-HMG-CoA did not inhibit P. mevalonii HMG-CoA lyase.

Acyl Coenzyme A↗

PubMed1991

3-Hydroxy-3-methylglutaryldithio-coenzyme A: a potent inhibitor of Pseudomonas mevalonii HMG-CoA reductase.

3-Hydroxy-3-methyl-1-thionoglutaryl-coenzyme A, a dithioester analog of 3-hydroxy-3-methylglutaryl-CoA, has been enzymatically synthesized using the HMG-CoA synthase catalyzed condensation of acetyl-CoA with 3-oxo-1-thionobutyryl-CoA. HMGdithio-CoA is a potent inhibitor of Pseudomonas mevalonii HMG-CoA reductase. Inhibition was mainly competitive with respect to HMG-CoA with a Kis of 0.086 +/- .01 microM and noncompetitive with respect to NADH with a Kis of 3.7 +/- 1.5 microM and a Kii of 0.65 +/- .05 microM in the presence of 110 microM (R.S)-HMG-CoA.

Acyl Coenzyme A↗

PubMed1991

The cis-acting regulatory element of the mvaAB operon of Pseudomonas mevalonii.

DNA upstream of the transcription start site of the mvaAB operon of Pseudomonas mevalonii, which encodes 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.88) and HMG-CoA lyase (EC 4.1.3.4), contains a cis-acting regulatory element which functions in the response to mevalonate. The regulatory element resides within a 36-bp region located from 48 to 84 bp upstream of the transcription start site of mvaA. This location was inferred from the beta-galactosidase activities of P. mevalonii harboring plasmid-encoded mvaA-lacZ fusions induced by mevalonate and by DNA gel retardation and competition assays. While protein from P. mevalonii grown on mevalonate produced a band shift, protein from cells grown on succinate gave no band shift, even when mevalonate was added. The operator contains three 10-bp direct repeats with the consensus sequence TGGGTACAGT, which may be important for regulation of the mvaAB operon.

Base Sequence↗

PubMed1990

Identification of the principal catalytically important acidic residue of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

Kinetic analysis of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase has implicated a glutamate or aspartate residue in (i) formation of mevaldate thiohemiacetal by proton transfer to the carbonyl oxygen of mevaldate and (ii) enhanced ionization of CoASH by the resulting enzyme carboxylate anion, facilitating attack by CoAS- on the carbonyl carbon of mevaldate (Veloso, D., Cleland, W. W., and Porter, J. W. (1981) Biochemistry 81, 887-894). Although neither the identity of this acidic residue nor its location is known, the catalytic domains of 11 sequenced HMG-CoA reductases contain only 3 conserved acidic residues. For HMG-CoA reductase of Pseudomonas mevalonii, these residues are Glu52, Glu83, and Asp183. To identify the acidic residue that functions in catalysis, we generated mutants having alterations in these residues. The mutant proteins were expressed, purified, and characterized. Mutational alteration of residues Glu52 or Asp183 of P. mevalonii HMG-CoA reductase yielded enzymes with significant, but in some cases reduced, activity (Vmax = 100% Asp183----Ala, 65% Asp183----Asn, and 15% Glu52----Gln of wild-type activity, respectively). Although the activity of mutant enzymes Glu52----Gln and Asp183----Ala was undetectable under standard assay conditions, their Km values for substrates were 4-300-fold higher than those for wild-type enzyme. Km values for wild-type enzyme and for mutant enzymes Glu52----Gln and Asp183----Ala were, respectively: 0.41, 73, and 120 mM [R,S)-mevalonate); 0.080, 4.4, and 2.0 mM (coenzyme A); and 0.26, 4.4, and 1.0 mM (NAD+). By these criteria, neither Glu52 nor Asp183 is the acidic catalytic residue although each may function in substrate recognition. During chromatography on coenzyme A agarose or HMG-CoA agarose, mutant enzymes Asp183----Asn and Glu83----Gln behaved like wild-type enzyme. By contrast, and in support of a role for these residues in substrate recognition, mutant enzymes Glu52----Gln and Asp183----Ala exhibited impaired ability to bind to either support. Despite displaying Km values for substrates and chromatographic behavior on substrate affinity supports comparable to wild-type enzyme, only mutant enzyme Glu83----Gln was essentially inactive under all conditions studied (Vmax = 0.2% that of wild-type enzyme). Glutamate residue 83 of P. mevalonii HMG-CoA reductase, and consequently the glutamate of the consensus Pro-Met-Ala-Thr-Thr-Glu-Gly-Cys-Leu-Val-Ala motif of the catalytic domains of eukaryotic HMG-CoA reductases, is judged to be the acidic residue functional in catalysis.

Amino Acid Sequence↗

PubMed1989

Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-CoA reductase. Characterization and chemical modification.

Pseudomonas mevalonii (formerly designated Pseudomonas sp. M (Beach, M. J., and Rodwell, V. W. (1989) J. Bacteriol. 171, 2994-3001; Gill, J. F., Jr., Beach, M.J., and Rodwell, V. W. (1985) J. Biol. Chem. 260, 9393-9398] 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.88), overexpressed in Escherichia coli (1), has been purified to electrophoretic homogeneity in 75% yield (final specific activity 48 mumols of NAD+ reduced per min/mg protein). The enzyme catalyzes its normal catabolic reaction (mevalonate + 2 NAD+ + CoASH----HMG-CoA + 2NADH + 2H+), and two half-reactions which involve mevaldehyde, the postulated intermediate in the aforementioned reactions and mevaldehyde + NADH + H+----mevalonate + NAD+). The rates of all four reactions and the Michaelis constants for all substrates were measured. Coenzyme A decreased the KM for mevaldehyde reduction 12-fold and stimulated VMAX 2-3 fold. CoASH thus may remain bound throughout the catalytic cycle. Dithiothreitol and analogs of CoASH were tested for their ability to reproduce the CoASH stimulation. Pantetheine, but not dithiothreitol, pantothenate, or desulfo-CoA mimicked CoASH stimulation. Titration with 5,5'-dithiobis(2-nitrobenzoic acid) indicated two sulfhydryl groups per subunit. Both groups remained accessible to 5,5'-dithiobis(2-nitrobenzoic acid) in the presence of mevalonate and/or NAD+ but only one group in the presence of HMG-CoA. N-Ethylmaleimide inhibited all the aforementioned reactions. HMG-CoA, but not mevalonate, afforded protection completely and irreversibly inactivated the enzyme. The reactive sulfhydryl group thus may not be a catalytic residue, but may be involved in a conformational change.

Chromatography, Ion Exchange↗

PubMed1989

Role of cysteine residues in Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-CoA reductase. Site-directed mutagenesis and characterization of the mutant enzymes.

Each of the four identical subunits of Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase contains two cysteine residues, Cys156 and Cys296 (Beach, M. J., and Rodwell, V. W. (1989) J. Bacteriol. 171, 2994-3001). Both are accessible to modification by sulfhydryl reagents under nondenaturing conditions (Jordan-Starck, T. C., and Rodwell, V. W. (1989) J. Biol. Chem. 264, 17913-17918). We used site-directed mutagenesis to construct three mutant enzymes in which alanine replaced either or both cysteine residues. Mutant enzymes C156A, C296A, and C156/296A were over-expressed in Escherichia coli and were found to be fully active. Following their purification, all four forms of the enzyme were compared with respect to their catalytic efficiency, their affinities for the substrates of all four catalyzed reactions, and for their sensitivity to inactivation by sulfhydryl reagents. Replacement of cysteine residues with alanine residues had no major effect on either the specific activity or the affinity of the enzymes for any substrate. The mutants catalyzed all four HMG-CoA reductase reactions as efficiently as did the wild-type enzyme, and coenzyme A stimulated mevaldehyde reduction to the same extent as for wild-type HMG-CoA reductase. Mutant C156A and the cysteine-free mutant C156/296A were not inactivated by 5,5'-dithiobis(2-nitrobenzoate). By contrast, mutant C296A was inactivated to the same extent as was the wild-type enzyme. Following treatment of the mutant enzymes with N-ethylmaleimide, the four reductase reactions catalyzed by mutant C296A were inactivated to the same extent as for the wild-type enzyme. Neither mutant C156A nor C156/296A was affected by this reagent. We conclude that the sulfhydryl reagent-reactive group whose derivatization leads to loss of enzymatic activity is Cys156. However, this residue is not an essential active site residue since neither substrate binding nor catalysis was affected when it was replaced by alanine. Possible roles of cysteine in maintaining structural stability are discussed.

Amino Acid Sequence↗

PubMed1989

3-Hydroxy-3-methylglutaryl coenzyme A lyase from Pseudomonas mevalonii.

HMG-CoA lyase, the putative second intracellular enzyme of mevalonate catabolism in Pseudomonas mevalonii (which we previously referred to as Pseudomonas sp. M (Gill et al. (1984) J. Bacteriol. 160, 294-298, Gill et al. (1985) J. Biol. Chem. 250, 9393-9398 and Sherban, M.S., Thesis, Purdue University), was purified 650-fold from cell extracts to a specific activity of 22 mumol acetyl-CoA formed per min per mg protein. This represents the first published report of the partial purification and characterization of an HMG-CoA lyase from a prokaryotic source. Cleavage of HMG-CoA produced acetyl-CoA and acetoacetate. Activity was optimal at pH 8.8 and was undetectable at or below pH 6.5. The estimated Km for S-HMG-CoA was 100 microM. Both a reduced thiol and Mg2+ or Mn2+ were required for activity. While Mn2+ was preferred at low concentrations, 1 mM or higher concentrations of either cation supported the same maximum velocity. An apparent native Mr of 40,400 +/- 3100 was estimated from gel filtration and sucrose density gradient ultracentrifugation data.

Cations, Divalent↗

PubMed1989

(S)-3-hydroxy-3-methylglutaryl coenzyme A reductase, a product of the mva operon of Pseudomonas mevalonii, is regulated at the transcriptional level.

We have cloned and sequenced a 505-base-pair (bp) segment of DNA situated upstream of mvaA, the structural gene for (S)-3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.88) of Pseudomonas mevalonii. The DNA segment that we characterized includes the promoter region for the mva operon. Nuclease S1 mapping and primer extension analysis showed that mvaA is the promoter-proximal gene of the mva operon. Transcription initiates at -56 bp relative to the first A (+1) of the translation start site. Transcription in vivo was induced by mevalonate. Structural features of the mva promoter region include an 80-bp A + T-rich region, and -12, -24 consensus sequences that resemble sequences of sigma 54 promoters in enteric organisms. The relative amplitudes of catalytic activity, enzyme protein, and mvaA mRNA are consistent with a model of regulation of this operon at the transcriptional level.

Base Sequence↗

PubMed1989

Nucleotide sequence and expression in Escherichia coli of the 3-hydroxy-3-methylglutaryl coenzyme A lyase gene of Pseudomonas mevalonii.

The mva operon of Pseudomonas mevalonii encodes two enzymes that can convert internalized mevalonate into acetoacetate and acetyl-coenzyme A (CoA). The promoter-proximal gene of this operon is mvaA, the structural gene for 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase (EC 1.1.1.88). The cloning, characterization, and expression of mvaA has been reported (M. J. Beach and V. W. Rodwell, J. Bacteriol. 171:2994-3001, 1989). We report here the nucleotide sequence of another gene of this operon, mvaB, its expression in Escherichia coli, and its identification as the structural gene for HMG-CoA lyase (EC 4.1.3.4). P. mevalonii HMG-CoA lyase is a cytosolic protein with 301 amino acid residues and a molecular weight of 31,600. This represents the first reported sequence of an HMG-CoA lyase from any source.

Amino Acid Sequence↗

PubMed1989

Cloning, sequencing, and overexpression of mvaA, which encodes Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl coenzyme A reductase.

We have cloned, determined the primary structure of, and overexpressed in Escherichia coli the gene mvaA, which is the 1,287-base structural gene for the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase [EC 1.1.1.88] of Pseudomonas mevalonii. The amino acid composition of HMG-CoA reductase agreed with that predicted from the nucleotide sequence of mvaA, and DNA-derived sequences were identical to all experimentally determined peptide sequences. Overexpression of mvaA in E. coli yielded quantities of HMG-CoA reductase over 1,500-fold higher than those present in control cultures. Comparison of the primary structure of the P. mevalonii enzyme with the DNA-derived primary structure for a mammalian HMG-CoA reductase revealed two regions of similarity suggestive of functional relatedness. An open reading frame, ORF1, lies on the 3' side of mvaA, and a potential ribosome-binding site for ORF1 overlaps the termination codon of mvaA.

Amino Acid Sequence↗