Protein engineering of Pseudomonas mevalonii HMG-CoA reductase determinants of nucleotide coenzyme specificity and conversion to a form whose activity is regulated by phosphorylation/dephosphorylation

Jon Alan Friesen, Purdue University

Abstract

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (E.C. 1.1.1.88) from the soil bacterium Pseudomonas mevalonii, an enzyme not naturally regulated by phosphorylation, utilizes NAD$\sp+$ to oxidize mevalonate to HMG-CoA. By contrast, mammalian HMG-CoA reductases are regulated by phosphorylation and utilize NADPH to reduce HMG-CoA to mevalonate. P. mevalonii HMG-CoA reductase was engineered to confer the following properties: (1) Improved use of the unnatural coenzyme NADP$\sp+$ and (2) Regulation by reversible phosphorylation-dephosphorylation. The 550,000-fold preference of P. mevalonii HMG-CoA reductase for NAD$\sp+$ versus NADP$\sp+$ was reduced to just a 7-fold preference by simultaneously substituting alanine for aspartate 146, which appears to hydrogen-bond to the 2$\sp\prime$-hydroxyl of the adenine ribose of NAD$\sp+,$ and changing adjacent residue leucine148 to arginine. The activity of HMG-CoA reductases of higher eukarya is regulated by reversible phosphorylation of a putative active site serine. Since in P. mevalonii HMG-CoA reductase arginine387 replaces the phosphoacceptor serine, activity is not regulated by phosphorylation. An initial attempt to engineer forms of P. mevalonii HMG-CoA reductase whose activity is modulated by phosphorylation by constructing P. mevalonii-hamster chimeric enzymes yielded chimera that were phosphorylatable but inactive. Arginine387 was then replaced by serine, generating an active enzyme, but one that was not readily phosphorylated. Multiple amino acid residues around serine387 were next mutated, revealing combinations of amino acids critical for phosphorylation of HMG-CoA reductase by HMG-CoA reductase kinase. This generated two active mutant enzymes into which phosphate could be incorporated whose activity was reversibly modulated by phosphorylation-dephosphorylation. This appears to represent the only known instance where an enzyme has been engineered to a form whose activity can be modulated by phosphorylation-dephosphorylation. That the reversible phosphorylation models mammalian resembles that of the mammalian enzyme. The structure of a P. mevalonii HMG-CoA reductase whose activity is modulated by incorporation and release of phosphate should ultimately provide a structural view of the mechanism whereby the activity of HMG-CoA reductase is attenuated by phosphorylation.

Degree

Ph.D.

Advisors

Rodwell, Purdue University.

Subject Area

Biochemistry|Molecular biology

COinS