Engineering of Sulfolobus solfataricus HMG-CoA reductase to a form whose activity is regulated by phosphorylation and dephosphorylation

There are two classes of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) re ductase: the class I enzymes of eukaryotes and some archaea, and the class II enzymes of certain eubacteria. The activity of the class I Syrian hamste r HMG-CoA reductase is regulated by phosphorylation-dephosphorylation of Se r871. 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, lack ing. The HMG-CoA reductase of the thermophilic archaeon Sulfolobus solfatar icus, whose stability recommends it for physical studies, lacks both a phos phoacceptor serine and a protein kinase recognition motif. Consequently, it s activity is not regulated by phosphorylation. We therefore employed site- directed mutagenesis to engineer an appropriately located phosphoacceptor s erine 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 LA03 R/G404R/A406S. The general properties of enzyme L403R/G404R/A406S (K-m valu es, V-max, optimal pH and temperature) were essentially those of the wild-t ype enzyme. Exposure of enzyme L403R/G404R/A406S to [gamma-P-32]ATP and cAM P-dependent protein kinase was accompanied by incorporation of P-32(i) and by a parallel decrease in catalytic activity. Subsequent treatment with a p rotein phosphatase released enzyme-bound P-32(i) and restored activity to p retreatment levels. The regulatory properties of enzyme LA03R/G404R/A406S t hus match those of the hamster enzyme. Solution of the three-dimensional st ructures of the phospho and dephospho forms of this mutant enzyme thus shou ld reveal structural features critical for regulation of the activity of a class I HMG-CoA reductase.