Kurosaki F
Laboratory of Cell Biology, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Japan, USA.
Arch Biochem Biophys. 1995 Aug 1;321(1):239-44. doi: 10.1006/abbi.1995.1391.
6-Hydroxymellein synthase, an inducible polyketide biosynthetic enzyme in carrot cell extracts, is composed of two identical subunits, and the homodimer is dissociated to monomeric peptides under high-ionic-strength conditions with loss of the synthase activity. Appreciable radioactivities were associated with the synthase proteins when the monomer enzyme was incubated with the radiolabeled substrates, acetyl-coenzyme A (CoA) and malonyl-CoA. Therefore, it appeared that the synthase does not lose the ability of binding the substrate even after the dissociation to monomers. The monomeric synthase liberated triacetic acid lactone as the derailment product instead of 6-hydroxymellein from the enzyme-attached triketomethylene chain which is the immediate precursor of an NADPH-dependent keto-reducing reaction involved in 6-hydroxymellein biosynthesis. These observations strongly suggest that the monomeric synthase retains the ability of ketomethylene chain elongation by the condensation of acyl-CoAs, but is lacking in an NADPH-dependent keto-reducing activity toward the triketide intermediate. Results obtained in the present experiments imply that the catalytic domain of acyl-CoA condensation is able to associate with that of keto reduction, possibly belonging to another subunit, only in the homodimeric structure to organize the multicatalytic reaction center.
6-羟基蜜白霉素合酶是胡萝卜细胞提取物中一种可诱导的聚酮生物合成酶，由两个相同的亚基组成，在高离子强度条件下，同型二聚体解离为单体肽，同时合酶活性丧失。当单体酶与放射性标记的底物乙酰辅酶A（CoA）和丙二酰辅酶A一起孵育时，合酶蛋白上出现了明显的放射性。因此，即使解离为单体后，合酶似乎也没有丧失结合底物的能力。单体合酶从与酶相连的三酮亚甲基链上释放出三乙酸内酯作为脱轨产物，而不是6-羟基蜜白霉素，三酮亚甲基链是6-羟基蜜白霉素生物合成中依赖NADPH的酮还原反应的直接前体。这些观察结果强烈表明，单体合酶保留了通过酰基辅酶A缩合进行酮亚甲基链延长的能力，但对三酮中间体缺乏依赖NADPH的酮还原活性。本实验获得的结果表明，酰基辅酶A缩合的催化结构域能够与酮还原的催化结构域结合，后者可能属于另一个亚基，仅在同型二聚体结构中才能组织多催化反应中心。
