Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, SK, Canada S7N 5C9.
Bioorg Med Chem. 2011 Feb 15;19(4):1390-9. doi: 10.1016/j.bmc.2011.01.011. Epub 2011 Jan 13.
Brassinin oxidase, a fungal detoxifying enzyme that mediates the conversion of the phytoalexin brassinin into indole-3-carboxaldehyde, is the first enzyme described to date that catalyzes the transformation of a dithiocarbamate group into an aldehyde equivalent. Brassinin is an essential phytoalexin due to its antifungal activity and its role as biosynthetic precursor of other phytoalexins produced in plants of the family Brassicaceae (common name crucifer). In this report, the isolation, structure determination and synthesis of the elusive co-product of brassinin transformation by brassinin oxidase, S-methyl dithiocarbamate, the syntheses of dideuterated and (R) and (S) monodeuterated brassinins, kinetic analyses of isotope effects and chemical modifications of brassinin oxidase are described. The reaction of [1'-(2)H(2)]brassinin was found to be slowed by a kinetic isotope effect of 5.3 on the value of k(cat)/K(m). This result indicates that the hydride/hydrogen transfer step preceding brassinin transformation is rate determining in the overall reaction. In addition, the use of (R) and (S)-[1'-(2)H]brassinins as substrates indicated that the hydride/hydrogen transfer step is ca. 88% stereoselective for the pro-R hydrogen. A detailed chemical mechanism of the enzymatic transformation of brassinin is proposed.
菜豆素氧化酶，一种真菌解毒酶，介导植物抗毒素菜豆素向吲哚-3-甲醛的转化，是迄今为止描述的第一种催化二硫代氨基甲酸盐基团转化为醛等价物的酶。菜豆素是一种必需的植物抗毒素，因为它具有抗真菌活性，并且是十字花科植物中产生的其他植物抗毒素的生物合成前体（通用名 crucifer）。在本报告中，描述了菜豆素氧化酶转化菜豆素的难以捉摸的副产物 S-甲基二硫代氨基甲酸盐、氘代和（R）和（S）单氘代菜豆素的分离、结构确定和合成、同位素效应的动力学分析以及菜豆素氧化酶的化学修饰。发现[1'-(2)H(2)]菜豆素的反应被 k(cat)/K(m)值的动力学同位素效应 5.3 减慢。这一结果表明，在整个反应中，氢化物/氢转移步骤是菜豆素转化的速率决定步骤。此外，使用（R）和（S）-[1'-(2)H]菜豆素作为底物表明，氢化物/氢转移步骤对 pro-R 氢的立体选择性约为 88%。提出了菜豆素酶促转化的详细化学机制。
