MEVALONATE CATABOLISM IN PSEUDOMONAS SP. M (INDUCTION, TRANSPORT, HMG-COENZYME-A REDUCTASE)

JOHN FRANCIS GILL, Purdue University

Abstract

Pseudomonas sp. M has a previously undescribed inducible transport system for mevalonate. This high affinity (K(,m) = 88 (mu)M for R,S-mevalonate) system was energy-dependent and highly substrate specific. Pseudomonas sp. M HMG-CoA reductase, the first enzyme of mevalonate catabolism, was purified 23-fold in 54% yield (> 15 mg of protein from 4 liters of cells) to > 99% homogeneity (final specific activity 60,500 nmol NAD('+) reduced/min/mg protein). R-mevalonate was the substrate. Enzyme activity was sensitive to sulfhydryl modifying reagents. The molecular weight of the holoenzyme was 178,000 Da (gel filtration chromatography) and that of the subunit was 43,000 Da (SDS-polyacrylamide gels). The enzyme thus appears to be a tetramer. Comparison of a 23 residue amino terminal sequence obtained for Pseudomonas HMG-CoA reductase with that from Chinese Hamster Ovary cell HMG-CoA reductase showed little homology. Antibody raised against Pseudomonas HMG-CoA reductase failed to recognize rat liver HMG-CoA reductase. Thus, little structural homology appears to exist between Pseudomonas and mammalian HMG-CoA reductases. Growth on mevalonate induced HMG-CoA reductase activity 200 to 800-fold and mevalonate transport activity 40 to 65-fold. Addition of mevalonate to starved cells produced a 2 hour lag in the induction of HMG-CoA reductase activity which preceded the induction of mevalonate transport activity by 2 hours. Induction of both activities preceded the initiation of cell growth and thus was required for growth on mevalonate. Addition of mevalonate to cells actively growing on glucose caused a more rapid (within 30 min) and biphasic pattern of induction of both HMG-CoA reductase activity and mevalonate transport. A biphasic pattern of cell growth was also observed under these conditions. Glucose was the preferred carbon source during phase I of growth and mevalonate during phase II. Nearly 30% of the preferred mevalonate isomer was depleted from the medium during phase I without significant metabolism to CO(,2). Mevalonate or a mevalonate catabolite thus may accumulate in cells of Pseudomonas sp. M during phase I and glucose metabolism may inhibit or repress the expression of a later catabolic enzyme. Under these conditions, accumulated acetoacetate might act as a feedback inhibitor of HMG-CoA reductase since acetoacetate inhibited HMG-CoA reductase competitively with respect to mevalonate.

Degree

Ph.D.

Subject Area

Microbiology

COinS