Structure-Function Relationship of Vibrio harveyi NADPH-Flavin Oxidoreductase FRP: Essential Residues Lys167 and Arg15 for NADPH Binding
Structure-Function Relationship of Vibrio harveyi NADPH-Flavin Oxidoreductase FRP: Essential Residues Lys167 and Arg15 for NADPH Binding
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DOI：
10.1021/bi3002314
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发表时间：
2012-06-19
期刊：
影响因子：
2.9
通讯作者：
Tu, Shiao-Chun
中科院分区：
文献类型：
--
作者：
Chung, Hae-Won;Tu, Shiao-Chun
哈维氏弧菌NADPH-FMN氧化还原酶（FRP）催化NADPH还原黄素。在与大肠杆菌NfsA比较氨基酸序列和晶体结构时，靶向残基N134、R225、R133、K167和R15以研究它们在NADPH底物的结合和利用中的可能作用。通过将这五个残基中的每一个突变为丙氨酸，稳态速率分析表明，变体K167 A和R15 A具有明显大大增加的K-m，K-NADPH和降低的k（cat）/K-m，K-NADPH，而对于其它变体发现很少或更温和的变化。K167 A和R15 A的（4 R）-[4-H-2]-NADPH的氘同位素效应（D）（V/K）分别显著增加至6.3和7.4，表明NADPH和NADP（+）解离的速率常数相对于氢化物转移步骤大大增强。此外，厌氧停流分析显示，K167 A和R15 A的NADPH结合平衡解离常数（Kd）分别为2.5-3.9和1.1 mM，远高于天然FRP的0.4 μ M Kd，而这两种变体的k（cat）与野生型酶相似。此外，K167到丙氨酸的突变甚至导致NADH的k（cat）/K-m的轻微增加。综合考虑，这些结果为以下结论提供了强有力的支持：K167和R15在FRP与NADPH的结合中均至关重要。这种功能作用也可能存在于其他FRP同源蛋白。
Vibrio harveyi NADPH-FMN oxidoreductase (FRP) catalyzes flavin reduction by NADPH. In comparing amino acid sequence and crystal structure with Escherichia coli NfsA, residues N134, R225, R133, K167, and R15 were targeted for investigation of their possible roles in the binding and utilization of the NADPH substrate. By mutation of each of these five residues to an alanine, steady-state rate analyses showed that the variants K167A and R15A had apparently greatly increased K-m,K-NADPH and reduced k(cat)/K-m,K-NADPH, whereas little or much more modest changes were found for the other variants. The deuterium isotope effects (D)(V/K) for (4R)-[4-H-2]-NADPH were markedly increased to 6.3 and 7.4 for K167A and R15A, respectively, indicating that the rate constants for NADPH and NADP(+) dissociation were greatly enhanced relative to the hydride transfer steps. Also, anaerobic stopped-flow analyses revealed that the equilibrium dissociation constant for NADPH binding (K-d) to be 2.5-3.9 and 1.1 mM for K167A and R15A, respectively, much higher than the 0.4 mu M K-d for the native FRP, whereas the k(cat) of these two variants were similar to that of the wild-type enzyme. Moreover, the K167 to alanine mutation led to even a slight increase in k(cat)/K-m for NADH. These results, taken together, provide a strong support to the conclusion that K167 and R15 each was critical in the binding of NADPH by FRP. Such a functional role may also exist for other FRP homologous proteins.