Partial purification and characterization of a polyketide biosynthetic enzyme, 6-hydroxymellein synthase, in elicitor-treated carrot cell extracts.
6-Hydroxymellein synthase, an induced polyketide biosynthetic enzyme in carrot cell extracts, was purified about 240-fold and its properties were compared with those of fatty acid synthetases. Synthetic activity of 6-hydroxymellein was inhibited in the presence of sulfhydryl reagents; however, cerulenin, a well-known inhibitor of fatty acid synthetases, showed no inhibitory activity to the enzyme. Biosynthesis of 6-hydroxymellein includes an NADPH-dependent ketoreduction, and, in this reaction, the 4-pro-S-hydrogen of NADPH was specifically transferred to the compound. On the basis of stereochemical analyses of the biosynthetic process, it was concluded that the product of the ketoreduction is an optically active alcohol of R configuration. These stereo-specificities of the reduction process are identical to those of beta-ketoacyl reductase in fatty acid biosynthesis which are considered to be conserved in all organisms. The synthetic rate of 6-hydroxymellein was markedly reduced when the assay was carried out with deuterium-labeled NADPH. The observed isotope effect on the catalytic rate (kH/kD) was 5.20, suggesting that this ketoreduction is one of the rate-limiting processes in 6-hydroxymellein synthesis. More than 85% of the synthetic activity was found in the soluble fraction of carrot cells, and, unlike in fatty acid synthetases in higher plants, organelle-localizing activity was not observed.
Kurosaki
F
F
Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Japan.
Itoh
M
M
Kizawa
Y
Y
Nishi
A
A
D000217:Acyltransferases / Q000302:isolation & purification* / Q000378:metabolism*; D002569:Cerulenin / Q000494:pharmacology; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D064429:Fatty Acid Synthases / Q000378:metabolism*; D007700:Kinetics; D008025:Ligases / Q000302:isolation & purification* / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000302:isolation & purification* / Q000378:metabolism*; D010088:Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism*; D010944:Plants / Q000201:enzymology*; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D009097:Multienzyme Complexes; D013439:Sulfhydryl Reagents; D002569:Cerulenin; D010088:Oxidoreductases; D000217:Acyltransferases; D064429:Fatty Acid Synthases; C070623:6-hydroxymellein synthetase; D008025:Ligases