Background l-Fucose is a rare sugar with potential uses in the pharmaceutical, cosmetic, and food industries. The enzymatic approach using l-fucose isomerase, which interconverts l-fucose and l-fuculose, can be an efficient way of producing l-fucose for industrial applications. Here, we performed biochemical and structural analyses of l-fucose isomerase identified from a novel species of Raoultella (RdFucI). Results RdFucI exhibited higher enzymatic activity for l-fuculose than for l-fucose, and the rate for the reverse reaction of converting l-fuculose to l-fucose was higher than that for the forward reaction of converting l-fucose to l-fuculose. In the equilibrium mixture, a much higher proportion of l-fucose (~ ninefold) was achieved at 30 °C and pH 7, indicating that the enzyme-catalyzed reaction favors the formation of l-fucose from l-fuculose. When biochemical analysis was conducted using l-fuculose as the substrate, the optimal conditions for RdFucI activity were determined to be 40 °C and pH 10. However, the equilibrium composition was not affected by reaction temperature in the range of 30 to 50 °C. Furthermore, RdFucI was found to be a metalloenzyme requiring Mn2+ as a cofactor. The comparative crystal structural analysis of RdFucI revealed the distinct conformation of α7-α8 loop of RdFucI. The loop is present at the entry of the substrate binding pocket and may affect the catalytic activity. Conclusions RdFucI-catalyzed isomerization favored the reaction from l-fuculose to l-fucose. The biochemical and structural data of RdFucI will be helpful for the better understanding of the molecular mechanism of l-FucIs and the industrial production of l-fucose.

中文翻译：

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使用来自 Raoultella sp. 的岩藻糖异构酶从 l-岩藻糖酶促合成 l-岩藻糖。以及酶的生化和结构分析。

背景 l-岩藻糖是一种稀有糖，在制药、化妆品和食品行业具有潜在用途。使用 l-岩藻糖异构酶的酶促方法可相互转化 l-岩藻糖和 l-岩藻糖，是生产用于工业应用的 l-岩藻糖的有效方法。在这里，我们对从一种新的 Raoultella (RdFucI) 物种中鉴定出的 l-岩藻糖异构酶进行了生化和结构分析。结果 RdFucI 对 l-岩藻糖的酶活性高于对 l-岩藻糖的酶活性，将 l-岩藻糖转化为 l-岩藻糖的逆反应速率高于将 l-岩藻糖转化为 l-岩藻糖的正反应速率。在平衡混合物中，在 30 °C 和 pH 7 下，L-岩藻糖的比例更高（约九倍），表明酶催化反应有利于从 l-岩藻糖形成 l-岩藻糖。当以 l-岩藻糖为底物进行生化分析时，确定 RdFucI 活性的最佳条件为 40 °C 和 pH 10。但在 30 至 50 °C 范围内，平衡组成不受反应温度的影响. 此外，发现 RdFucI 是一种需要 Mn2+ 作为辅助因子的金属酶。RdFucI 的比较晶体结构分析揭示了 RdFucI 的 α7-α8 环的独特构象。该环存在于底物结合口袋的入口处，可能会影响催化活性。结论 RdFucI 催化的异构化有利于从 l-岩藻糖到 l-岩藻糖的反应。