Expression and characterization of the HMG-CoA reductase of the thermophilic archaeon Sulfolobus solfataricus.
The thermostable class I HMG-CoA reductase of Sulfolobus solfataricus offers potential for industrial applications and for the initiation of crystallization trials of a biosynthetic HMG-CoA reductase. However, of the 15 arginine codons of the hmgA gene that encodes S. solfataricus HMG-CoA reductase, 14 (93%) are AGA or AGG, the arginine codons used least frequently by Escherichia coli. The presence of these rare codons in tandem or in the first 20 codons of a gene can complicate expression of that gene in E. coli. Problems include premature chain termination and misincorporation of lysine for arginine. We therefore sought to improve the expression and subsequent yield of S. solfataricus HMG-CoA reductase by expanding the pool size of tRNA(AGA,AGG), the tRNA that recognizes these two rare codons. Coexpression of the S. solfataricus hmgA gene with the argU gene that encodes tRNA(AGA,AGG) resulted in an over 10-fold increase in enzyme yield. This has provided significantly greater quantities of purified enzyme for potential industrial applications and for crystallographic characterization of a stable class I HMG-CoA reductase. It has, in addition, facilitated determination of kinetic parameters and of pH optima for all four catalyzed reactions, for determination of the K(i) for inhibition by the statin drug mevinolin, and for comparison of the properties of the HMG-CoA reductase of this thermophilic archaeon to those of other class I HMG-CoA reductases.
Kim
D Y
DY
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907, USA.
Bochar
D A
DA
Stauffacher
C V
CV
Rodwell
V W
VW
D000214:Acyl Coenzyme A / Q000378:metabolism; D000215:Acylation; D002847:Chromatography, Agarose; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D006903:Hydroxymethylglutaryl CoA Reductases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D007700:Kinetics; D008148:Lovastatin / Q000494:pharmacology; D008798:Mevalonic Acid / Q000378:metabolism; D012347:RNA, Transfer, Arg / Q000235:genetics / Q000378:metabolism; D016944:Sulfolobus / Q000201:enzymology*
D000214:Acyl Coenzyme A; D012347:RNA, Transfer, Arg; C008047:3-hydroxy-3-methylglutaryl-coenzyme A; D008148:Lovastatin; D006903:Hydroxymethylglutaryl CoA Reductases; D008798:Mevalonic Acid