3-Hydroxy-3-methylglutaryl CoA reductase and mevalonate kinase of Neurospora crassa.
Two enzymes of polyisoprenoid synthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase (mevalonate:NADP oxidoreductase [acylating CoA], EC 1.1.1.34) and mevalonate kinase (ATP:mevalonate 5-phosphotransferase, EC 2.7.1.36), are present in the microsomal and soluble fractions of Neurospora crassa, respectively. HMG CoA reductase specifically uses NADPH as reductant and has a K(m) for dl-HMG CoA of 30 micro M. The activities of HMG CoA reductase and mevalonate kinase are low in conidia and increase threefold during the first 12 hr of stationary growth. Maximum specific activities of both enzymes occur when aerial hyphae and conidia first appear (2 days), but total activities peak later (3-4 days). Addition to the growth media of ergosterol or beta-carotene, alone or in combination, does not affect the specific or total activity of either enzyme. The mevalonate kinase of N. crassa, purified 200-fold to a specific activity of 5 micro moles/min/mg, is free from HMG CoA reductase, phosphomevalonate kinase, ATPase, adenylate kinase, and NADH oxidase activities. Mevalonate kinase specifically requires ATP as cosubstrate and exhibits a marked preference for Mg(2+) over Mn(2+), especially at high ratios of divalent metal ion to ATP. Kinase activity is inhibited by p-hydroxymercuribenzoate, and this inhibition is partially prevented by mevalonate or MgATP. Optimum activity occurs at pH 8.0-8.5 and at about 55 degrees C. The Neurospora kinase, like that of hog liver, has a sequential mechanism for substrate addition. The Michaelis constants obtained were 2.8 mM for dl-mevalonate and 1.8 mM for MgATP(-2). Geranyl pyrophosphate is an inhibitor competitive with MgATP (K(i) = 0.11 mM).
D000255:Adenosine Triphosphate; D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D002250:Carbon Radioisotopes; D002338:Carotenoids / Q000494:pharmacology; D002413:Cations, Divalent; D002455:Cell Division; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002855:Chromatography, Thin Layer; D004875:Ergosterol / Q000494:pharmacology; D005977:Glutarates; D006863:Hydrogen-Ion Concentration; D006902:Hydroxymercuribenzoates / Q000494:pharmacology; D007700:Kinetics; D008798:Mevalonic Acid; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D010770:Phosphotransferases / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D013347:Subcellular Fractions / Q000201:enzymology; D013696:Temperature; D013997:Time Factors
D002250:Carbon Radioisotopes; D002413:Cations, Divalent; D005977:Glutarates; D006902:Hydroxymercuribenzoates; D002338:Carotenoids; D000255:Adenosine Triphosphate; D000429:Alcohol Oxidoreductases; D010770:Phosphotransferases; D008798:Mevalonic Acid; D004875:Ergosterol