<br>DesII 的表征和机理研究：参与 TDP-d-去糖胺生物合成的自由基 S-腺苷-L-甲硫氨酸酶,Journal of the American Chemical Society

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DesII 的表征和机理研究：参与 TDP-d-去糖胺生物合成的自由基 S-腺苷-L-甲硫氨酸酶
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2009-10-07 , DOI: 10.1021/ja903354k
Ping-Hui Szu ₁ , Mark W Ruszczycky , Sei-hyun Choi , Feng Yan , Hung-wen Liu

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D-desosamine (1) 是一种 3-(N,N-二甲氨基)-3,4,6-三脱氧己糖，存在于多种大环内酯类抗生素中，包括甲霉素 (2)、新甲霉素 (3)、匹克霉素 (4) 和那博霉素(5)由委内瑞拉链霉菌产生。它在赋予其母体苷元生物活性方面发挥着重要作用。先前对委内瑞拉沙门氏菌中去糖胺生物合成的遗传和生化研究表明，TDP-4-氨基-4,6-二脱氧-D-葡萄糖 (8) 转化为 TDP-3-酮-4,6-二脱氧- D-葡萄糖 (9) 由 DesII 催化，DesII 是自由基 S-腺苷-L-甲硫氨酸 (SAM) 酶超家族的成员。在这里，我们报告了 His(6) 标记 DesII 的纯化和重建，使用 UV-vis 和 EPR 光谱表征其 [4Fe-4S] 簇，以及黄素氧还蛋白、黄素氧还蛋白还原酶和 NADPH 还原 [4Fe-4S] 簇的能力。 -4S](2+)簇。还包括 DesII 催化反应的稳态动力学分析以及 DesII 底物灵活性的研究。使用 TDP-[3-(2)H]-4-氨基-4,6-二脱氧-D-葡萄糖作为底物将氘掺入 SAM 的研究为氢原子直接转移到 5'-脱氧腺苷自由基提供了强有力的证据。催化循环。 C-3 处发生氢原子夺取的事实也揭示了这种有趣的脱氨基反应的机制。

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Characterization and Mechanistic Studies of DesII: A RadicalS-Adenosyl-l-methionine Enzyme Involved in the Biosynthesis of TDP-d-Desosamine

D-desosamine (1) is a 3-(N,N-dimethylamino)-3,4,6-trideoxyhexose found in a number of macrolide antibiotics including methymycin (2), neomethymycin (3), pikromycin (4), and narbomycin (5) produced by Streptomyces venezuelae . It plays an essential role in conferring biological activities to its parent aglycones. Previous genetic and biochemical studies of the biosynthesis of desosamine in S. venezuelae showed that the conversion of TDP-4-amino-4,6-dideoxy-D-glucose (8) to TDP-3-keto-4,6-dideoxy-D-glucose (9) is catalyzed by DesII, which is a member of the radical S-adenosyl-L-methionine (SAM) enzyme superfamily. Here, we report the purification and reconstitution of His(6)-tagged DesII, characterization of its [4Fe-4S] cluster using UV-vis and EPR spectroscopies, and the capability of flavodoxin, flavodoxin reductase, and NADPH to reduce the [4Fe-4S](2+) cluster. Also included are a steady-state kinetic analysis of DesII-catalyzed reaction and an investigation of the substrate flexibility of DesII. Studies of deuterium incorporation into SAM using TDP-[3-(2)H]-4-amino-4,6-dideoxy-D-glucose as the substrate provides strong evidence for direct hydrogen atom transfer to a 5'-deoxyadenosyl radical in the catalytic cycle. The fact that hydrogen atom abstraction occurs at C-3 also sheds light on the mechanism of this intriguing deamination reaction.

更新日期：2009-10-07

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