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

Abstract

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.

Avian 3‐hydroxy‐3‐methylglutaryl‐CoA lyase: Sensitivity of enzyme activity to thiol/disulfide exchange and identification of proximal reactive cysteines

“…4B); similar results are obtained even when the bacterial enzyme is exposed to higher concentrations (20-fold excess with respect to enzyme protomer) of the modification reagent. Absence in the prokaryotic enzyme (Anderson & Rodwell, 1989) of a cysteine-containing peptide homologous to that targeted in the avian enzyme (Table 2) would appear compatible with these observations. …”

“…Therefore, it is likely that any thiol/disulfide exchange reaction identified in the avian enzyme also occurs in the human enzyme. However, it is interesting to note that the protein sequence deduced from analysis of P. mevalonii DNA that encodes HMG-CoA lyase (Anderson & Rodwell, 1989) does not indicate a comparable cysteine in this C-terminal region. The fact that this cysteine is not conserved clearly argues against a catalytically essential role for this residue.…”

“…Following hydroxylapatite chromatography, the enzyme was loaded onto a blue-Sepharose column equilibrated in 20 mM sodium phosphate, pH 7.0, containing 0.5 mM DTT and eluted with the same buffer containing 100 pM HMG-CoA. Pseudomonas mevalonii HMG-CoA lyase was isolated from E. coli containing the pT7-2600 plasmid reported by Anderson and Rodwell (1989) using the method of Narasimhan and Miziorko (1993). For assays with reduced HMG-CoA lyase, enzyme (350 U/mg) was preincubated in 40 mM Tris-C1, pH 8.2, with 40 mM DTT for >30 min at 30 "C. Following complete reduction, the enzyme was placed in an anaerobic chamber and rapidly passed over a Sepha-dex G-50 centrifugal desalting column (Penefsky, 1977) equilibrated in oxygen-free 100 mM potassium phosphate, pH 7.8.…”

A ColE1-encoded gene directs entry exclusion of the plasmid

“…In vivo protein synthesis was analyzed by a modification of the method of Anderson and Rodwell (2). BL21(DE3) cells harboring a pVEX11 recombinant plasmid and pLysS were grown at 37ЊC in LB supplemented with ampicillin (50 g/ml) and chloramphenicol (20 g/ml) to an A 600 of 0.5.…”

Identification of Genes and Proteins Necessary for Catabolism of Acyclic Terpenes and Leucine/Isovalerate in Pseudomonas aeruginosa

“…The cells of interest (2 to 3 g) were resuspended in 1 ml of 0.1 M HEPES buffer (pH 7.4) per g of cells before 100 l DNase I (100 g/ml), 50 l RNase A (10 mg/ml), and 150 l 10 mM MgSO 4 were added. The suspension was passed two times through a precooled French press cell at 800 2 and centrifuged at 80,000 ϫ g for 1 h at 4°C. 2D electrophoresis was performed using 18-cm-long immobilized pH gradient strips (pH 3 to 10 or pH 4 to 7) that had been rehydrated under mineral oil at room temperature overnight {340 l rehydration solution contained 7 M urea, 2 M thiourea, 2% (wt/vol) 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, 0.002% (wt/vol) bromphenol blue, 10 mM dithiothreitol (DTT), 0.5% Pharmalyte pH 3 to 10 or pH 5 to 8, and 500 g soluble protein}.…”