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Journal of Lipid Research, Vol. 15, 211-222, May 1974
Copyright © 1974 by Lipid Research, Inc.

3-Hydroxy-3-methylglutaryl CoA reductase and mevalonate kinase of Neurospora crassa

Richard L. Imblum and Victor W. Rodwell

Department of Biochemistry, Purdue University, Lafayette, Indiana 47907

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 µM. 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 ßbeta;-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 µmoles/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 Mg²⁺ over Mn²⁺, 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°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).

Supplementary key words biosynthesis of carotenoids • biosynthesis of sterols • biosynthesis of isoprenoids • comparative biochemistry of Neurospora

Submitted on May 4, 1973
Revised on December 4, 1973
Accepted on December 31, 1973

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