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3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Activity in Ochromonas malhamensis

A System to Study the Relationship between Enzyme Activity and Rate of Steroid Biosynthesis

Department of Chemistry, Portland State University, Portland, Oregon 97207, Biotechnology Group, Amoco Research Center, P.O. Box 400, Naperville, Illinois 60566

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the key regulatory enzyme of the isoprenoid pathway, was found to be predominantly microsomal in Ochromonas malhamensis, a chrysophytic alga. Detection of HMG-CoA reductase requires the presence of 1% bovine serum albumin during cell homogenization, and the activity is stimulated by the presence of Triton X-100. The enzyme has a pH optimum of 8.0 and an absolute requirement for NADPH. When grown in 10 micromolar mevinolin, a competitive inhibitor of HMG-CoA reductase, O. malhamensis shows a 10- to 15-fold increase in HMG-CoA reductase activity (after washing) with little or no effect on cell growth rate. Cultures can be maintained in 10 micromolar mevinolin for months. O. malhamensis produces a large amount (1% dry weight) of poriferasterol, a product of the isoprenoid pathway. The addition of 10 micromolar mevinolin initially blocked poriferasterol biosynthesis by >90%; within 2 days the rate of synthesis returned to normal levels. Immediately after mevinolin was washed from the 2-day culture, there was a transient 2.5-fold increase in the rate of poriferasterol biosynthesis. The rate of poriferasterol biosynthesis and the level of HMG-CoA reductase activity both fell to control levels within hours.