Cofactor Requirements for the Formation of Δ9-Unsaturated Fatty Acids in Mycobacterium phlei

The conversion of stearyl coenzyme A and palmityl coenzyme -1 to the A9 derivatives by a particulate enzyme from yeast requires molecular oxygen and a reduced pyridine nucleotide (1, 2).These cofactors are also necessary for the formation of monounsaturated fatty acids by rat liver preparations (3, 4) and for the formation of polyunsaturated fatty acids in cell-free &ems from both liver (5) and yeast (6).As reported in a preliminary note (7), the desaturation of fatty acyl coenzyme A esters to the A9 derivatives in cell-free preparations from Mycobacteriwn phki requires Fe++ and a flavin, in addition to reduced triphosphopyridine nucleotide and 02.We now wish to describe a more quantitative study of these cofactor requirements.EXPERINENTAL PROCEDURE Materials-Stearic-1-'"C and palmitic-I-*% acids were purchased from the New England Nuclear Corporation and were purified before use by treatment of the methyl esters with mercuric acetate followed by silicic acid chromatography (8) to remove any polar or unsaturated impurities.The fatty acids purified in this manner contained less than 0.1% unsaturated material and had a radiochemical purity greater than 97% as judged by gas-liquid and silicic acid chromatography.The Coh derivatives were prepared from the purified fatty acids by the procedure of Goldman and Vagelos (9).Tetrahydrofolic acid was prepared by catalytic hydrogenation of folic acid in glacial acetic acid (10).Dihydrolipoic acid was obtained by reduction of lipoic acid with NaBH4 in aqueous ethanol (11).2-Amino-4-hydroxy-6,7-dimethyltetrahydropteridine was the gift of Dr. Alisa Tietz.Phenazine methosulfate, catalase, and TPN+ were purchased from the Sigma Chemical Company, and all other cofactors and enzyme inhibitors were from the California Corporation for Biochemical Research.Activated silicic acid (Unisil, 100 to 200 mesh) was purchased from the Clarkson Chemical Company, Inc.Methods-M.phlei ATCC 356 was grown on a defined medium for 48 to 60 hours at 37" as previously described (7).Cells were harvested, washed by centrifugation, and suspended (180 mg per ml) in 0.25 M sucrose.This suspension was disrupted sonically for 8 minutes in 25-ml batches in a Raytheon model DF-IO1 sonic oscillator.