Characterization and mechanistic study of a radical SAM dehydrogenase in the biosynthesis of butirosin
Characterization and mechanistic study of a radical SAM dehydrogenase in the biosynthesis of butirosin
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DOI：
10.1021/ja072481t
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发表时间：
2007-12-12
影响因子：
15
通讯作者：
Eguchi, Tadashi
中科院分区：
文献类型：
--
作者：
Yokoyama, Kenichi;Numakura, Mario;Eguchi, Tadashi
Butirosin 生物合成基因簇中编码的 BtrN 具有在自由基 S-腺苷甲硫氨酸 (SAM) 超家族中保守的 CXXXCXXC 基序。它在布替罗辛生产者环状芽孢杆菌中的基因破坏导致 2-脱氧青蟹肌胺 (DOIA) 和 2-脱氧链霉胺 (DOS) 之间的生物合成途径中断。此外，过表达酶的体外测定表明，在严格厌氧条件下，BtrN 在消耗等摩尔量的 SAM 的情况下催化 DOIA 氧化，产生 5'-脱氧腺苷、蛋氨酸和 3-氨基-2,3-二脱氧-青蟹肌糖 (amino-DOI)。动力学分析显示 DOIA 抑制底物，而 SAM 不抑制底物，这表明该反应是有序 Bi Ter 机制，SAM 是第一个底物，DOIA 是第二个底物。 BtrN 与 [3-H-2]DOIA 的反应生成未标记的、单氘化和二氘化的 5'-脱氧腺苷，而通过在氧化氘缓冲液中孵育未标记的 DOIA，不会掺入氘。这些结果表明，DOIA C-3 上的氢原子直接转移到 5'-脱氧腺苷上，得到 DOIA 自由基中间体，生成未标记和双氘代的 5'-脱氧腺苷，证明了夺氢步骤的可逆性。目前的研究表明，BtrN 是一种不寻常的自由基 SAM 脱氢酶，通过自由基机制催化羟基的氧化。这是自由基SAM酶氧化羟基的机理研究的第一份报告。
BtrN encoded in the butirosin biosynthetic gene cluster possesses a CXXXCXXC motif conserved within the radical S-adenosyl methionine (SAM) superfamily. Its gene disruption in the butirosin producer Bacillus circulans caused the interruption of the biosynthetic pathway between 2-deoxy-scyllo-inosamine (DOIA) and 2-deoxystreptamine (DOS). Further, in vitro assay of the overexpressed enzyme revealed that BtrN catalyzed the oxidation of DOIA under the strictly anaerobic conditions along with consumption of an equimolar amount of SAM to produce 5'-deoxyadenosine, methionine, and 3-amino-2,3-dideoxy-scyllo-inosose (amino-DOI). Kinetic analysis showed substrate inhibition by DOIA but not by SAM, which suggests that the reaction is the Ordered Bi Ter mechanism and that SAM is the first substrate and DOIA is the second. The BtrN reaction with [3-H-2]DOIA generated nonlabeled, monodeuterated and dideuterated 5'-deoxyadenosines, while no deuterium was incorporated by incubation of nonlabeled DOIA in the deuterium oxide buffer. These results indicated that the hydrogen atom at C-3 of DOIA was directly transferred to 5'-deoxyadenosine to give the radical intermediate of DOIA Generation of nonlabeled and dideuterated 5'-deoxyadenosines proved the reversibility of the hydrogen abstraction step. The present study suggests that BtrN is an unusual radical SAM dehydrogenase catalyzing the oxidation of the hydroxyl group by a radical mechanism. This is the first report of the mechanistic study on the oxidation of a hydroxyl group by a radical SAM enzyme.