Identification of elements critical for phosphorylation of 3-hydroxy-3-methylglutaryl coenzyme A reductase by adenosine monophosphate-activated protein kinase: protein engineering of the naturally nonphosphorylatable 3-hydroxy-3-methylglutaryl coenzyme A reductase from Pseudomonas mevalonii.

The initially nonphosphorylatable 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase of Pseudomonas mevalonii (E.C. 1.1.1.88) was engineered to phosphorylatable forms in order to identify elements critical for phosphorylation of HMG-CoA reductase by AMP-activated protein kinase. P. mevalonii, mutant enzymes phosphorylatable by AMP-activated protein kinase were engineered by substituting cognate residues from the kinase recognition sequence of Syrian hamster HMG-CoA reductase (E.C. 1.1.1.34). Various combinations of residues 381-391, which correspond to the kinase recognition sequence of the hamster enzyme, were mutated. P. mevalonii mutant enzyme R387S, in which a serine had been inserted at position P, which corresponds to that of the regulatory serine of the hamster enzyme, was only weakly phosphorylated. Genes that encoded thirty-six additional mutant enzymes containing various portions of the hamster kinase recognition sequence were constructed. Following expression, purified mutant enzymes were assayed as substrates for AMP-activated protein kinase. Identified as critical for phosphorylation was the simultaneous presence of aspartate or asparagine at position P+3 and of leucine at position P+4, three and four residues on the C-terminal side of the phosphorylatable serine, respectively. Two basic residues at positions P-1, P-2, or P-3 also appeared to be critical for phosphorylation when present in combination with aspartate or asparagine at P+3 and leucine at P+4.