Flavin mononucleotide (FMN)-dependent ene-reductases constitute a large family of oxidoreductases that catalyze the enantiospecific reduction of carbon–carbon double bonds. The reducing equivalents required for substrate reduction are obtained from reduced nicotinamide by hydride transfer. Most ene-reductases significantly prefer, or exclusively accept, either NADPH or NADH. Despite their usefulness in biocatalytic applications, the structural determinants for cofactor preference remain elusive. We employed the NADPH-preferring 12-oxophytodienoic acid reductase 3 from Solanum lycopersicum (SlOPR3) as a model enzyme of the ene-reductase family and applied computational and structural methods to investigate the binding specificity of the reducing coenzymes. Initial docking results indicated that the arginine triad R283, R343, and R366 residing on and close to a critical loop at the active site (loop 6) are the main contributors to NADPH binding. In contrast, NADH binds unfavorably in the opposite direction toward the β-hairpin flap within a largely hydrophobic region. Notably, the crystal structures of SlOPR3 in complex with either NADPH₄ or NADH₄ corroborated these different binding modes. Molecular dynamics simulations confirmed NADH binding near the β-hairpin flap and provided structural explanations for the low binding affinity of NADH to SlOPR3. We postulate that cofactor specificity is determined by the arginine triad/loop 6 and the residue(s) controlling access to a hydrophobic cleft formed by the β-hairpin flap. Thus, NADPH preference depends on a properly positioned arginine triad, whereas granting access to the hydrophobic cleft at the β-hairpin flap favors NADH binding.

中文翻译：

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环 6 和 β-发夹瓣是决定 FMN 依赖性烯还原酶家族中辅因子特异性的结构热点

黄素单核苷酸（FMN）依赖性烯还原酶构成了一个氧化还原酶大家族，可催化​​碳-碳双键的对映特异性还原。底物还原所需的还原当量是通过氢化物转移从还原的烟酰胺获得的。大多数烯还原酶明显偏爱或完全接受 NADPH 或 NADH。尽管它们在生物催化应用中很有用，但辅助因子偏好的结构决定因素仍然难以捉摸。我们采用来自番茄的 NADPH 偏好的 12-氧代植物二烯酸还原酶 3 ( Sl OPR3) 作为烯还原酶家族的模型酶，并应用计算和结构方法来研究还原辅酶的结合特异性。初步对接结果表明，位于活性位点关键环（环 6）上及其附近的精氨酸三联体 R283、R343 和 R366 是 NADPH 结合的主要贡献者。相反，NADH 在主要疏水区域内以相反方向不利地结合到 β-发夹瓣。值得注意的是， Sl OPR3 与 NADPH ₄或 NADH ₄复合的晶体结构证实了这些不同的结合模式。分子动力学模拟证实了 NADH 在 β-发夹瓣附近结合，并为 NADH 与Sl OPR3 的低结合亲和力提供了结构解释。我们假设辅因子特异性由精氨酸三联体/环 6 和控制进入由 β-发夹瓣形成的疏水裂口的残基决定。因此，NADPH 的偏好取决于正确定位的精氨酸三联体，而允许进入 β-发夹瓣处的疏水裂口有利于 NADH 结合。