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.

Phosphorylation of a UDP-glucuronosyltransferase regulates substrate specificity

“…Another possibility is that UGT catalysis, which requires partially negatively charged (nucleophilic) acceptor substrates (e.g., eugenol and 17␤-estradiol) (9), utilizes a phosphothreonine͞serine operationally to enhance proton abstraction (25,26) from hydroxyl or carboxyl group(s) in the numerous substrates with wide structural variations. Such an effect could enhance the interaction of an acceptor substrate with partially positively charged carbon-1 of the common donor substrate, UDP-glucuronic acid.…”

Debottlenecking of an In Vitro Enzyme Cascade Using a Combined Model‐ and Experiment‐Based Approach

“…Supporting Information for this article can be found under DOI: https://doi.org/10.1002/cite.202200170. This section includes additional references to primary literature relevant for this research [20][21][22][23][24][25][26][27][28].…”

Diversity in domain architectures of Ser/Thr kinases and their homologues in prokaryotes

“…The phosphorylated proteins undergo changes in their three-dimensional (3-D) shapes resulting in altered functional states. Phosphorylation events also lead to changes in the electrostatic interactions and ionisation states of residues at the catalytic site as exemplified by regulation of isocitrate dehydrogenases and HMG-CoA reductase [1,2]. Covalent attachment of the phosphoryl groups to various proteins is catalysed by protein kinases.…”