Division of Medicinal Chemistry, College of Pharmacy, and Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, Texas 78712, USA.
J Am Chem Soc. 2010 Feb 24;132(7):2359-69. doi: 10.1021/ja909451a.
DesII from Streptomyces venezuelae is a radical SAM (S-adenosyl-l-methionine) enzyme that catalyzes the deamination of TDP-4-amino-4,6-dideoxy-d-glucose to form TDP-3-keto-4,6-dideoxy-d-glucose in the biosynthesis of TDP-d-desosamine. DesII also catalyzes the dehydrogenation of the nonphysiological substrate TDP-D-quinovose to TDP-3-keto-6-deoxy-d-glucose. These properties prompted an investigation of how DesII handles SAM in the redox neutral deamination versus the oxidative dehydrogenation reactions. This work was facilitated by the development of an enzymatic synthesis of TDP-4-amino-4,6-dideoxy-d-glucose that couples a transamination equilibrium to the thermodynamically favorable oxidation of formate. In this study, DesII is found to consume SAM versus TDP-sugar with stoichiometries of 0.96 +/- 0.05 and 1.01 +/- 0.05 in the deamination and dehydrogenation reactions, respectively, using Na(2)S(2)O(4) as the reductant. Importantly, no significant change in stoichiometry is observed when the flavodoxin/flavodoxin NADP(+) oxidoreductase/NADPH reducing system is used in place of Na(2)S(2)O(4). Moreover, there is no evidence of an uncoupled or abortive process in the deamination reaction, as indicated by the observation that dehydrogenation can take place in the absence of an external source of reductant whereas deamination cannot. Mechanistic and biochemical implications of these results are discussed.
委内瑞拉链霉菌 DesII 是一种依赖于 SAM（S-腺苷甲硫氨酸）的酶，能够催化 TDP-4-氨基-4,6-二脱氧-d-葡萄糖发生脱氨反应，生成 TDP-3-酮-4,6-二脱氧-d-葡萄糖，这是 TDP-d-去甲涎糖胺生物合成途径中的一步。DesII 还能催化非生理底物 TDP-D-奎诺糖发生脱氢反应，生成 TDP-3-酮-6-脱氧-d-葡萄糖。这些特性促使我们研究 DesII 在还原中性脱氨反应和氧化脱氢反应中如何处理 SAM。本研究通过开发一种 TDP-4-氨基-4,6-二脱氧-d-葡萄糖的酶促合成方法得以实现，该方法通过转氨平衡将反应热力学有利的甲酸氧化耦联起来。本研究发现，DesII 在脱氨和脱氢反应中分别以 0.96 ± 0.05 和 1.01 ± 0.05 的化学计量比消耗 SAM 与 TDP-糖，其中还原剂为 Na(2)S(2)O(4)。重要的是，当使用黄素蛋白/黄素蛋白 NADP(+)氧化还原酶/NADPH 还原系统替代 Na(2)S(2)O(4)时，化学计量比没有明显变化。此外，在脱氨反应中没有观察到无偶联或无效的过程，因为在没有外部还原剂的情况下可以进行脱氢反应，而脱氨反应则不能。讨论了这些结果在机制和生化方面的意义。
