Pseudomonas mevalonii HMG-CoA reductase: Transcriptional regulation and identification of a glutamate essential for catalysis

Yuli Wang, Purdue University

Abstract

Pseudomonas mevalonii HMG-CoA reductase (E.C. 1.1.1.38) is induced in cells grown on mevalonate (J. F. Gill, Jr., M. J. Beach, and V. W. Rodwell, J. Bacteriol. 160, 294-298, 1984), and the mvaA gene, which encodes P. mevalonii HMG-CoA reductase, has been cloned and sequenced (M. J. Beach and V. W. Rodwell, J. Bacteriol. 171, 2994-3001, 1989). Measurements of HMG-CoA reductase activity, protein, and mRNA revealed that induction is controlled at the transcriptional level. The transcription initiation site is 56 bp upstream of the adenosine of the ATG translation start codon of mvaA. The mvaA promoter has $-$12 and $-$24 consensus sequences typical of prokaryotic promoters which utilize sigma$\sp{54}$ RNA polymerase holoenzyme and activator proteins for transcription. The cis-acting element which responds to mevalonate was shown, by assays of the expression of mva-lacZ translation fusions and by DNA gel retardation assays, to be on a 36 pb DNA segment 48 pb upstream of the transcription initiation site. From kinetic data, D. Veloso, W. W. Cleland, and J. W. Porter (Biochemistry 20, 887-894, 1981) inferred that an acidic residue functions in catalysis by yeast HMG-CoA reductase. The catalytic domains of 11 HMG-CoA reductases contain 3 conserved acidic residues. These 3 residues of P. mevalonii HMB-CoA reductase were changed to glutamine (E52 and E83), asparagine (D183), or alanine (D183) by site-directed mutagenesis. All four mutant enzymes were then overexpressed, purified, and characterized. V$\sb{\rm max}$ for mutant enzymes E52Q, D183A, and D183N was 15, 69, and 109% that of wild-type HMG-CoA reductase, respectively. Thus, neither Glu$\sp{52}$ nor Asp$\sp{183}$ is a catalytic residue. Mutant enzymes E52Q and D183A had elevated K$\sb{\rm m}$ values for all substrates and impaired ability to bind to coenzyme A or HMG-CoA affinity supports. By contrast, mutant enzyme E83Q had a V$\sb{\rm max}$ value only 0.4% that of wild-type HMG-CoA reductase. This low value appeared to result neither from an altered conformation nor from an impaired inability to bind substrates. For mutant enzyme E83Q, K$\sb{\rm m}$ values for NAD$\sp{+}$, R,S-mevalonate, and coenzyme A, and chromatography on affinity supports was typical of wild-type enzyme. The data are consistent with residue E83 of P. mevalonii HMG-CoA reductase being the acidic residue functional in catalysis.

Degree

Ph.D.

Advisors

Rodwell, Purdue University.

Subject Area

Biochemistry

COinS