Characterization of an HMG-CoA reductase from Listeria monocytogenes that exhibits dual coenzyme specificity.
HMG-CoA reductase (HMGR) is an enzyme critical for cellular cholesterol synthesis in mammals and isoprenoid synthesis in certain eubacteria, catalyzing the NAD(P)H-dependent reduction of HMG-CoA to mevalonate. We have isolated the gene encoding HMG-CoA reductase from Listeria monocytogenes and expressed the recombinant 6x-His-tagged form in Escherichia coli. Using NAD(P)(H), the enzyme catalyzes HMG-CoA reduction approximately 200-fold more efficiently than mevalonate oxidation in vitro. The purified enzyme exhibits dual coenzyme specificity, utilizing both NAD(H) and NADP(H) in catalysis; however, catalytic efficiency using NADP(H) is approximately 200 times greater than when using NAD(H). The statins mevinolin and mevastatin are weak inhibitors of L. monocytogenes HMG-CoA reductase, requiring micromolar concentrations for inhibition. Three-dimensional modeling reveals that the overall structure of L. monocytogenes HMG-CoA reductase is likely similar to the known structure of the class II enzyme from Pseudomonas mevalonii. It appears that the enzyme has catalytic amino acids in analogous positions that likely play similar roles and also has a flap domain that brings a catalytic histidine into the active site. However, in L. monocytogenes HMG-CoA reductase histidine 143 and methionine 186 are present in the putative NAD(P)(H)-selective site, possibly interacting with the 2' phosphate of NADP(H) or 2' hydroxyl of NAD(H) and providing the active site architecture necessary for dual coenzyme specificity.
Theivagt
Amy E
AE
Department of Chemistry, Illinois State University, Normal, Illinois 61790, USA.
Amanti
Elise N
EN
Beresford
Nicola J
NJ
Tabernero
Lydia
L
Friesen
Jon A
JA
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics; D002384:Catalysis; D045782:Hemiterpenes / Q000096:biosynthesis; D006801:Humans; D006903:Hydroxymethylglutaryl CoA Reductases / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008089:Listeria monocytogenes / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009943:Organophosphorus Compounds; D010455:Peptides / Q000737:chemistry / Q000235:genetics; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D000596:Amino Acids; D045782:Hemiterpenes; D009943:Organophosphorus Compounds; D010455:Peptides; C004809:isopentenyl pyrophosphate; D006903:Hydroxymethylglutaryl CoA Reductases