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T Shimakata

Publications and source records attributed to T Shimakata.

23 records · Page 2Linked to original sources
PubMed1980

Involvement of one of two enoyl-CoA hydratases and enoyl-CoA reductase in the acetyl-CoA-dependent elongation of medium chain fatty acids by Mycobacterium smegmatis.

2-Enoyl-CoA reductase was purified 150-fold from the crude extract of Mycobacterium smegmatis. The purified reductase required NADH, but not NADPH, as a reductant and catalyzed the reduction of C4 to C16 enoyl-CoAs, though the activities toward shorter chain substrates (c4 and C6) were very low. Thiolase was also partially purified from the same source. These two enzymes were used, together with 3-hydroxyacyl-CoA dehydrogenase and the two forms of enoyl-CoA hydratase (hydratases I and II) previously purified from the same source, to reconstitute fatty acid elongation activity. The products formed from [1-14C]acetyl-CoA and decanoyl-CoA in this reconstituted system were analyzed by thin-layer chromatography and radio-gas-liquid chromatography. The system containing hydratase II produced laurate and 3-hydroxylaurate (in the form of their CoA esters) and the ratio of laurate to 3-hydroxylaurate increased as the incubation time was increased. The system containing hydratase I produced only 3-hydroxylaurate. 3-Hydroxylaurate was also the only product when enoyl-CoA reductase was omitted from the system containing hydratase II. It is concluded that hydratase II, but not hydratase I, is functional in fatty acid elongation by M. smegmatis and that enoyl-CoA reductase is also essential for the reaction. CoA and NAD+ inhibited the reconstituted elongation activity in competition with acetyl-CoA and NADH, respectively.

Acetyl Coenzyme A↗
PubMed1979

Purification and characterization of 3-hydroxyacyl-CoA dehydrogenase of Mycobacterium smegmatis.

3-Hydroxyacyl-CoA dehydrogenase [EC 1.1.1.35] was purified 100-fold to homogeneity from crude extracts of Mycobacterium smegmatis, using ammonium sulfate fractionation, gel filtration, and chromatography on DEAE-cellulose, hydroxyapatite, and NAD-Sepharose 4B columns. Its molecular weight was estimated to be 50,300 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. NADH acted twelve times more efficiently than NADPH as an electron donor for the reduction of 3-ketoacyl-CoA, and there was strict substrate stereospecificity (L form) in the oxidation of 3-hydroxyacyl-CoA. The pH optimum depended upon the direction of reaction, i.e., 6.0 for the oxidation of NADH and 9--10 for the reduction of NAD. The Km values for different thioesters of acetoacetate, i.e., esters of CoA, pantetheine, and acetyl-cysteamine were determined to be 0.036, 1.19, and 44.4 mM, respectively. Antibodies raised against the dehydrogenase of M. smegmatis strongly inhibited the enzyme activity, but did not affect the corresponding dehydrogenase of pig heart. The antibodies were found to inhibit the acetyl-CoA dependent elongation of fatty acids by the crude extract of M. smegmatis. These findings, together with those on the reconstitution of the elongation activity reported previously (Shimakata, T., Fujita, Y., & Kusaka, T. (1977) J. Biochem. 82, 725-732) indicate that 3-hydroxyacyl-CoA dehydrogenase is involved in the acetyl-CoA dependent elongation of fatty acids in M. smegmatis.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1977

Acetyl-CoA-dependent elongation of fatty acids in Mycobacterium smegmatis.

An enzyme system of Mycobacterium smegmatis catalyzing the elongation of medium-chain fatty acids with acetyl-CoA was obtained free from de novo fatty acid synthetase by ammonium sulfate fractionation. The system was resolved by gel filtration and DEAE-cellulose chromatography into three fractions, all of which were required for reconstitution of the elongation activity. The three fractions were highly purified enoyl-CoA hydratase, highly purified 3-hydroxyacyl-CoA dehydrogenase, and a fraction containing both enoyl-CoA reductase and thiolase. The reconstituted system was avidin-insenstive, required NADH as a sole hydrogen donor, and was sensitive to pCMB, but not to N-ethylmaleimide or monoiodoacetate. Decanoyl-CoA and octanoyl-CoA were the best primers for the elongation system. When decanoyl-CoA was used as the primer, the major product was found to be a lauroyl derivative (probably lauroyl-CoA). Evidence was obtained suggesting that acyl-CoA dehydrogenase, catalyzing the first step of beta-oxidation, was not functional in the elongation system.

Acetyl-CoA C-Acetyltransferase↗