Bacterial metabolism of mevalonic acid conversion to acetoacetate

Siddiqi, Majid; Rodwell, Victor W.

doi:10.1016/0006-291x(62)90246-2

Cited by 16 publications

(11 citation statements)

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“…While the biosynthetic role of mevalonate in mammalian cells has been well established, little is known about mevalonate utilization in bacteria. Several procaryotes capable of growth on mevalonate as their sole carbon source have, however, been isolated (3,8,9,23,24,27).…”

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

Anderson

Rodwell

1989

J Bacteriol

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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.

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

Anderson

Rodwell

1989

J Bacteriol

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“…Class II HMG-CoA reductase from P. mevalonii (pmHMGR) catalyzes the oxidative acylation of mevalonate to form HMG-CoA, which is further converted into acetoacetate [1][2][3] . Since P. mevalonii utilizes R-mevalonate as a carbon source, the oxidative acylation of mevalonate is the predominantly occurring reaction in pmHMGR, unlike other homologues of this enzyme that have been characterized in other prokaryotic and eukaryotic organisms that favor the reduction of HMG-CoA for isoprenoid biosynthesis [4][5][6] .…”

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pH dependent inhibition from ammonium ions in the Pseudomonas mevalonii HMG-CoA Reductase crystallization environment

Purohit

Steussy

Rosales

et al. 2020

Preprint

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KEYWORDS KEYWORDFull form HMG-CoA 3-hydroxy-3-methylglutaryl-CoACoA Coenzyme A NAD Nicotinamide Adenine Dinucleotide pmHMGR Pseudomonas mMevalonii HMG-CoA reductase ABSTRACT HMG-CoA reductase (Pseudomonas mevalonii) utilizes mevalonate, coenzyme A (CoA) and the cofactor NAD in a complex mechanism involving two hydride transfers with cofactor exchange, accompanied by large conformational changes by a 50 residue subdomain, to generate HMG-CoA. Details about this mechanism such as the conformational changes that allow intermediate formation, cofactor exchange and product release remain unknown. The formation of the proposed intermediates has also not been observed in structural studies with natural substrates.Having been 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. The enzyme has been shown to be catalytically active inside the crystal with dithio-HMG-CoA and NADH but curiously is found to be inactive in the reverse direction in the structure bound to mevalonate, CoA and NAD.To understand the factors limiting activity in the HMGR crystal with mevalonate, CoA and NAD, we studied the effect of crystallization components and pH on enzymatic activity. We observed a strong inhibition in the crystallization buffer and an increase in activity with increasing pH. We attribute this inhibitive effect to the presence of ammonium ions present in the crystal since inhibition is also observed with several other ammonium salt buffers. Additionally, the lack of inhibition was observed in the absence of ammonium. The effect of each ligand (mevalonate, CoA and NAD) on the rate of the enzymatic reaction in the crystallization environment was further investigated by measuring their Km in the crystallization buffer. The Km measurements indicate that the hydride transfer step between NAD and mevalonate is inhibited in the crystallization environment. To test this further, we solved a crystal structure of pmHMGR bound to the post-hydride transfer intermediate (mevaldehyde) and cofactor Coenzyme A. The resulting turnover with the formation of a thiohemiacetal indicated that the crystallization environment inhibited the oxidative acylation of mevalonate and the reaction intermediate mevaldyl-CoA.

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“…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.Several microorganisms can utilize the isoprenoid precursor mevalonate as a sole carbon source (4,13,16,49, 50,52 …”

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“…Several microorganisms can utilize the isoprenoid precursor mevalonate as a sole carbon source (4,13,16,49, 50,52 …”

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

Beach

Rodwell

1989

J Bacteriol

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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.Several microorganisms can utilize the isoprenoid precursor mevalonate as a sole carbon source (4,13,16,49, 50,52 MATERIALS AND METHODSBacterial strains, plasmids, bacteriophage, and culture conditions. Plasmid cloning and M13 propagation were conducted in E. coli JM103 (36), using M9 medium, L broth (34), B broth, or 2x YT medium (36). Phage lambda was propagated in E. coli Q359 (25) on NZYM medium (34). Plasmids pUC18 and pUC19, phage derivatives M13mpl8 and M13mpl9, and lambda EMBL4 have been previously described (14, 54). Plasmid pKK177-3, a shortened version of pKK223-3 (9) in which the remainder of the tet gene between the BamHI and PvuII sites has been deleted (J. Brosius, personal communication), was obtained from Scott Buckel, Carnegie-Mellon University, Pittsburgh, Pa.Purification and assay of P. mevalonii HMG-CoA reductase. HMG-CoA reductase activity was assayed and the enzyme was purified to homogeneity as described by Gill et al. (17).Reduction, alkylation, and cleavage of protein with cyanogen bromide. Purified HMG-CoA reductase (450 nmol) was dialyzed against 9% (vol/vol) formic acid, lyophilized, and suspended in reduction-alkylation buffer (130 mM Tris [pH 8.0], 6 M guanidine hydrochloride, 1 mg of EDTA per ml) that contained dithioerythritol in a fourfold molar excess over the predicted sulfhydryl content. The solution was incubated at room temperature for 4 h and then dialyzed against reduction-alkylation buffer. Cysteine residues were alkylated by treatment first with 2 pCi of [1-14C]iodoacetamide (specific activity, 53 Ci/mol) and then with nonradioactive iodoacetamide. (Use of [14C]iodoacetamide was intended to facilitate identification of cysteine-containing peptides. However, no peptide yielded enough sequence to ascertain the positions of the two cysteine residues.) After 30 min, excess dithioerythritol was added to neutralize unreacted iodoacetamide. The reduced, alkylated protein was then dialyzed three times against 9% (vol/vol) formic acid, lyophilized, and suspended in 70% (vol/vol) formic acid. A crystal of cyanogen bromide was added, and cleavage was...

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Bacterial metabolism of mevalonic acid conversion to acetoacetate

Cited by 16 publications

References 10 publications

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

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

pH dependent inhibition from ammonium ions in the Pseudomonas mevalonii HMG-CoA Reductase crystallization environment

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

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