title: 3-hydroxy-3-methylglutaryl coenzyme A reductase of Sulfolobus solfataricus: DNA sequence, phylogeny, expression in Escherichia coli of the hmgA gene, and purification and kinetic characterization of the gene product.
abstract: The gene (hmgA) for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) from the thermophilic archaeon Sulfolobus solfataricus P2 was cloned and sequenced. S. solfataricus HMG-CoA reductase exhibited a high degree of sequence identity (47%) to the HMG-CoA reductase of the halophilic archaeon Haloferax volcanii. Phylogenetic analyses of HMG-CoA reductase protein sequences suggested that the two archaeal genes are distant homologs of eukaryotic genes. The only known bacterial HMG-CoA reductase, a strictly biodegradative enzyme from Pseudomonas mevalonii, is highly diverged from archaeal and eukaryotic HMG-CoA reductases. The S. solfataricus hmgA gene encodes a true biosynthetic HMG-CoA reductase. Expression of hmgA in Escherichia coli generated a protein that both converted HMG-CoA to mevalonate and cross-reacted with antibodies raised against rat liver HMG-CoA reductase. S. solfataricus HMG-CoA reductase was purified in 40% yield to a specific activity of 17.5 microU per mg at 50 degrees C by a sequence of steps that included heat treatment, ion-exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography. The final product was homogeneous, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The substrate was (S)- not (R)-HMG-CoA; the reductant was NADPH not NADH. The Km values for HMG-CoA (17 microM) and NADPH (23 microM) were similar in magnitude to those of other biosynthetic HMG-CoA reductases. Unlike other HMG-CoA reductases, the enzyme was stable at 90 degrees C and was optimally active at pH 5.5 and 85 degrees C.
authors.affiliation: Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA.
authors.forename: D A
authors.initials: DA
authors.lastname: Bochar
authors.forename: J R
authors.initials: JR
authors.lastname: Brown
authors.forename: W F
authors.initials: WF
authors.lastname: Doolittle
authors.forename: H P
authors.initials: HP
authors.lastname: Klenk
authors.forename: W
authors.initials: W
authors.lastname: Lam
authors.forename: M E
authors.initials: ME
authors.lastname: Schenk
authors.forename: C V
authors.initials: CV
authors.lastname: Stauffacher
authors.forename: V W
authors.initials: VW
authors.lastname: Rodwell
mesh_terms: D000214:Acyl Coenzyme A / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010802:Phylogeny; D051381:Rats; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D016944:Sulfolobus / Q000201:enzymology / Q000235:genetics*
chemical_list: D000214:Acyl Coenzyme A; D004269:DNA, Bacterial; C008047:3-hydroxy-3-methylglutaryl-coenzyme A