HMG-CoA reductase: The active site lysine, the Sulfolobus solfataricus enzyme, and cloning of genes for two class II enzymes

Daniel Austin Bochar, Purdue University

Abstract

In eukaryotes and archaea 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase catalyzes an early, rate-limiting reaction in isoprenoid biosynthesis that in humans is the target of drugs that reduce serum cholesterol levels. The only available crystal structure is that of the biodegradative enzyme from Pseudomonas mevalonii. This structure allowed the identification of an active site lysine, lysine 267. Mutation of lysine 267 to alanine, arginine, aspartate, histidine, or cysteine was accompanied by loss of detectable activity. Introduction of the non-naturally occurring amino acid aminoethylcysteine resulted in full recovery of activity. The future characterization of this modified enzyme will be important in the investigation of the role of lysine 267 in catalysis. In an attempt to identify in the enzyme from Syrian hamster the cognate residue of P. mevalonii lysine 267, lysines 690, 691, and 734 were mutated to alanine. While sequence alignments suggest lysine 734 as the active site lysine, the mutagenesis results did not allow the unequivocal assignment of the cognate residue. While the three mutant enzyme had detectable activity, all appeared impaired for catalysis with specific activities for enzymes K690A, K691A, and K734A of 0.2%, 1.6%, and 0.16% wild-type activity. Solution of a crystal structure of a biosynthetic HMG-CoA reductase would allow the identification of the active site lysine. The gene ( hmgA) for HMG-CoA reductase of the archaeon Sulfolobus solfataricus P2 was cloned and sequenced. S. solfataricus hmgA encodes a biosynthetic enzyme that was purified in 40% yield (specific activity 17.5 μU/mg at 50°C). The substrates are (S)-HMG-CoA (Km 17 μM) and NADPH (Km 23 μM). The enzyme was stable at 90°C and optimally active at pH 5.5 and 85°C. The thermostability of this enzyme makes it a good candidate for crystallization. Comparison of 50 sequences revealed two classes of HMG-CoA reductase. The catalytic domain of the human enzyme and the enzyme from P. mevalonii are proposed as canonical sequences for Class I and Class II HMG-CoA reductases, respectively. Only one Class II enzyme has previously been cloned, the biodegradative HMG-CoA reductase from P. mevalonii. Two new, apparently biosynthetic, Class II HMG-CoA reductase genes were cloned, from the eubacterium and Borrelia burgdorferi from the archaeon Archaeoglobus fulgidus.

Degree

Ph.D.

Advisors

Rodwell, Purdue University.

Subject Area

Biochemistry|Microbiology

COinS