3-HYDROXY-3-METHYLGLUTARYL COENZYME-A REDUCTASE OF SULFOLOBUS-SOLFATARICUS - DNA-SEQUENCE, PHYLOGENY, EXPRESSION IN ESCHERICHIA-COLI OF THEHMGA GENE, AND PURIFICATION AND KINETIC CHARACTERIZATION OF THE GENE-PRODUCT

The gene (hmgA) for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) re ductase (EC 1.1.1.34) from the thermophilic archaeon Sulfolobus solfat aricus P2 was cloned and sequenced. S. solfataricus HMG-CoA reductase exhibited a high degree of sequence identity (47%) to the HMG-CoA redu ctase of the halophilic archaeon Haloferax volcanii. Phylogenetic anal yses of HMG-CoA reductase protein sequences suggested that the two arc haeal genes are distant homologs of eukaryotic genes. The only known b acterial HMG-CoA reductase, a strictly biodegradative enzyme from Pseu domonas mevalonii, is highly diverged from archaeal and eukaryotic HMG -CoA reductases. The S. solfataricus hmgA gene encodes a true biosynth etic HMG-CoA reductase. Expression of hmgA in Escherichia coli generat ed a protein that both converted HMG-CoA to mevalonate and cross-react ed with antibodies raised against rat liver HMG-CoA reductase. S. solf ataricus HMG-CoA reductase was purified in 40% yield to a specific act ivity of 17.5 mu U per mg at 50 degrees C by a sequence of steps that included heat treatment, ion-exchange chromatography, hydrophobic inte raction chromatography, and affinity chromatography. The final product was homogeneous, as judged by sodium dodecyl sulfate-polyacrylamide g el electrophoresis. The substrate was (S)- not (R)-HMG-CoA; the reduct ant was NADPH not NADH. The K-m values for HMG-CoA (17 mu M) and NADPH (23 mu M) were similar in magnitude to those of other biosynthetic HM G-CoA reductases. Unlike other HMG-CoA reductases, the enzyme was stab le at 90 degrees C and was optimally active at pH 5.5 and 85 degrees C .