Biosynthesis of (S)-HMG-CoA and purification and characterization of HMG-CoA reductase from the archaeon Haloferax volcanii

Kenneth Mark Bischoff, Purdue University

Abstract

Two methods have been developed for use in the continuing investigation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. A method which employs Pseudomonas mevalonii HMG-CoA reductase for the enzymic synthesis of (S)-HMG-CoA, the physiologic substrate of HMG-CoA reductase, was developed. Subsequently, (R)-HMG-CoA has been isolated from commercially available (R,S)-HMG-CoA by selective cleavage of the (S)-diastereomer using P. mevalonii HMG-CoA lyase. (R)-HMG-CoA was shown to inhibit HMG-CoA reductase competitively with respect to the (S)-diastereomer, with a $\rm K\sb{i}/K\sb{m}$ ratio of about unity. Since the (R)-diastereomer is not an inert component of (R,S)-HMG-CoA, kinetic studies of HMG-CoA reductase should employ the enzymically synthesized substrate. The method described provides an economically favorable alternative to commercially available (R,S)-HMG-CoA. Second, a method for the purification of a new form of HMG-CoA reductase, that from the archaeon Haloferax volcanii was developed. The archaeal enzyme was soluble, utilized NADPH as cofactor and was halophilic, requiring 3 M KCl for optimal activity. The gene for Hf. volcanii HMG-CoA reductase was expressed in Escherichia coli and the enzyme was purified to electrophoretic homogeneity. Homogeneous Hf. volcanii HMG-CoA reductase had a specific activity of 24 $\mu$U/mg protein and catalyzed the half-reactions involving exogenously added mevaldehyde, a putative enzyme-bound intermediate, at rates comparable to Syrian hamster HMG-CoA reductase. Mutagenesis of the archaeal enzyme identified His398 as a catalytically essential residue. Addition of an asparate at position 404, 6 residues C-terminal of the catalytic histidine, attenuated activity, a result similar to the effect of phosphorylation of the hamster enzyme on Ser871, which also resides 6 residues from the catalytic histidine 865. The similarities between Hf. volcanii and hamster HMG-CoA reductases make the archaeal enzyme an attractive model for understanding the mechanism of phosphorylation induced attenuation of activity in the HMG-CoA reductases of higher eukaryotes.

Degree

Ph.D.

Advisors

Rodwell, Purdue University.

Subject Area

Biochemistry|Microbiology

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