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.
Friesen
J A
JA
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153, USA.
Rodwell
V W
VW
D055372:AMP-Activated Protein Kinases; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D006224:Cricetinae; D006801:Humans; D006903:Hydroxymethylglutaryl CoA Reductases / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008647:Mesocricetus; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017354:Point Mutation; D015202:Protein Engineering; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D011549:Pseudomonas / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine
D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D012694:Serine; D006903:Hydroxymethylglutaryl CoA Reductases; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases