title: Engineering of Sulfolobus solfataricus HMG-CoA reductase to a form whose activity is regulated by phosphorylation and dephosphorylation.
abstract: There are two classes of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase: the class I enzymes of eukaryotes and some archaea, and the class II enzymes of certain eubacteria. The activity of the class I Syrian hamster HMG-CoA reductase is regulated by phosphorylation-dephosphorylation of Ser871. Phosphorylation apparently prevents the active site histidine, His865, from protonating the inhibitory coenzyme A thioanion prior to its release from the enzyme. Structural evidence for this hypothesis is, however, lacking. The HMG-CoA reductase of the thermophilic archaeon Sulfolobus solfataricus, whose stability recommends it for physical studies, lacks both a phosphoacceptor serine and a protein kinase recognition motif. Consequently, its activity is not regulated by phosphorylation. We therefore employed site-directed mutagenesis to engineer an appropriately located phosphoacceptor serine and cAMP-dependent protein kinase recognition motif. Substitution of serine for Ala406, the apparent cognate of hamster Ser871, and replacement of Leu403 and Gly404 by arginine created S. solfataricus mutant enzyme L403R/G404R/A406S. The general properties of enzyme L403R/G404R/A406S (K(m) values, V(max), optimal pH and temperature) were essentially those of the wild-type enzyme. Exposure of enzyme L403R/G404R/A406S to [gamma-(32)P]ATP and cAMP-dependent protein kinase was accompanied by incorporation of (32)P(i) and by a parallel decrease in catalytic activity. Subsequent treatment with a protein phosphatase released enzyme-bound (32)P(i) and restored activity to pretreatment levels. The regulatory properties of enzyme L403R/G404R/A406S thus match those of the hamster enzyme. Solution of the three-dimensional structures of the phospho and dephospho forms of this mutant enzyme thus should reveal structural features critical for regulation of the activity of a class I HMG-CoA reductase.
authors.affiliation: Departments of Biochemistry and Biological Sciences, Purdue University, West Lafayette, Indiana 47907, USA.
authors.forename: D Y
authors.initials: DY
authors.lastname: Kim
authors.forename: C V
authors.initials: CV
authors.lastname: Stauffacher
authors.forename: V W
authors.initials: VW
authors.lastname: Rodwell
mesh_terms: D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics; D002384:Catalysis; D006224:Cricetinae; D004789:Enzyme Activation / Q000235:genetics; D005822:Genetic Vectors / Q000138:chemical synthesis; D006903:Hydroxymethylglutaryl CoA Reductases / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008647:Mesocricetus; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017354:Point Mutation; D016944:Sulfolobus / Q000201:enzymology* / Q000235:genetics*
chemical_list: D001224:Aspartic Acid; D006903:Hydroxymethylglutaryl CoA Reductases; D000409:Alanine