Thermostable archaeal HMG -CoA reductases: Regulation of the activity of Sulfolobus solfataricus HMG-CoA reductase by reversible phosphorylation and dual coenzyme specificity of Archaeoglobus fulgidus HMG-CoA reductase

Dong-Yul Kim, Purdue University

Abstract

The activity of the HMG-CoA reductases of higher eukaryotes is regulated by reversible phosphorylation of a putative active site serine. Mutagenic and kinetic evidence suggested that interaction between phosphoserine and the catalytic histidine, negates the ability of the histidine to protonate the departing coenzyme A thioanion. However, physical evidence for this interaction is lacking. The HMG-CoA reductase of the thermophilic archaeon Sulfolobus solfataricus contains alanine (Ala406) in place of a phosphorylatable serine. Its activity thus is not regulated by phosphorylation. In addition to providing long-term stability (retains activity above 80°C), a feature desirable both for physical studies and for possible exploitation as an industrial biocatalyst, this enzyme therefore represents a good candidate for crystallographic investigation. A form of S. solfataricus HMG-CoA reductase whose activity was regulated by reversible phosphorylation was engineered by replacing Ala406 by serine and introducing a cAMP-dependent protein kinase recognition motif. Ultimate solution of the three-dimensional structures of its phospho- and dephosphoforms could therefore reveal structural features critical for the phosphorylation-mediated regulation of HMGCoA reductase activity. The inferred amino acid sequence of orf AF1736 of Archaeoglobus fulgidus suggested that it might encode a Class II HMG-CoA reductase. Following PCR-based cloning of AF1736 from A. fulgidus genomic DNA and expression in Escherichia coli, the encoded enzyme was purified to apparent homogeneity and its enzymic properties were determined. Activity was optimal at 85°C. Mevinolin inhibited competitively with HMG-CoA. Unlike any other HMG-CoA reductase, the A. fulgidus enzyme exhibits dual coenzyme specificity. pH-activity profiles for all four catalyzed reactions revealed that optimal activity using NADP(H) occurred at a pH 1 to 3 units more acidic than that using NAD(H). Kinetic parameters were determined for all four reactions using either NAD(H) or NADP(H). These coenzymes compete for a common site. kcat[NAD(H)]/k cat[NADP(H)] varied from 1 to under 70 for the four reactions, indicative of a slight preference for NAD(H). Km and kcat values as a function of pH suggested that the protonated forms of two residues, probably His390 and Lys277, are essential for activity.

Degree

Ph.D.

Advisors

Rodwell, Purdue University.

Subject Area

Biochemistry

COinS