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

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Abstract
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.

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