Enzyme-catalyzed cofactor regeneration is a significant approach to avoid large quantities consumption of oxidized cofactor, which is vital in a variety of bioconversion reactions. NADH: FMN oxidoreductase is an ideal regenerating enzyme because innocuous molecular oxygen is required as an oxidant. But the by-product H2O2 limits its further applications at the industrial scale. Here, novel NADH: FMN oxidoreductase (LrFOR) from Lactobacillus rhamnosus comprised of 1146 bp with a predicted molecular weight of 42 kDa was cloned and overexpressed in Escherichia coli BL21 (DE3). Enzyme assay shows that the purified recombinant LrFOR has both the NADPH and NADH oxidation activity. Biochemical characterizations suggested that LrFOR exhibits the specific activity of 39.8 U·mg-1 with the optimal pH and temperature of 5.6 and 35 °C and produces H2O instead of potentially harmful peroxide. To further study its catalytic function, a critical Thr29 residue and its six mutants were investigated. Mutants T29G, T29A, and T29D show notable enhancement in activities compared with the wild type. Molecular docking of NADH into wild type and its mutants reveal that a small size or electronegative of residue in position29 could shorten the distance of NADH and FMN, promoting the electrons transfer and resulting in the increased activity. This work reveals the pivotal role of position 29 in the catalytic function of LrFOR and provides effective catalysts in NAD+ regeneration.

中文翻译：

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形成水的 NADH 的表达、生化特征和突变：鼠李糖乳杆菌的 FMN 氧化还原酶

酶催化辅因子再生是避免大量消耗氧化辅因子的重要方法，这在各种生物转化反应中至关重要。NADH：FMN 氧化还原酶是一种理想的再生酶，因为需要无害的分子氧作为氧化剂。但副产品 H2O2 限制了其在工业规模上的进一步应用。在这里，新型 NADH：来自鼠李糖乳杆菌的 FMN 氧化还原酶 (LrFOR) 由 1146 bp 组成，预测分子量为 42 kDa 被克隆并在大肠杆菌BL21 (DE3) 中过表达。酶学分析表明，纯化的重组LrFOR同时具有NADPH和NADH氧化活性。生化表征表明，LrFOR 在最适 pH 和温度为 5 时表现出 39.8 U·mg-1 的比活。6 和 35 °C 并产生 H2O 而不是潜在有害的过氧化物。为了进一步研究其催化功能，研究了一个关键的 Thr29 残基及其六个突变体。与野生型相比，突变体 T29G、T29A 和 T29D 的活性显着增强。NADH与野生型及其突变体的分子对接表明，29位残基的小尺寸或电负性可以缩短NADH与FMN的距离，促进电子转移并导致活性增加。这项工作揭示了 29 位在 LrFOR 催化功能中的关键作用，并为 NAD+ 再生提供了有效的催化剂。与野生型相比，T29D 的活性显着增强。NADH与野生型及其突变体的分子对接表明，29位残基的小尺寸或电负性可以缩短NADH与FMN的距离，促进电子转移并导致活性增加。这项工作揭示了 29 位在 LrFOR 催化功能中的关键作用，并为 NAD+ 再生提供了有效的催化剂。与野生型相比，T29D 的活性显着增强。NADH与野生型及其突变体的分子对接表明，29位残基的小尺寸或电负性可以缩短NADH与FMN的距离，促进电子转移并导致活性增加。这项工作揭示了 29 位在 LrFOR 催化功能中的关键作用，并为 NAD+ 再生提供了有效的催化剂。