X-Ray Crystallography of Flavin Reductase and Its Mutants

发光细菌的黄素还原酶与发光现象密切相关，硝基还原酶与突变密切相关。在本研究中，我们研究了野生型和突变型黄素还原酶(FRasel)和硝基还原酶(NFSA)。通过X射线结晶学研究。目的是在原子水平上阐明酶反应的机理和底物专一性。我们已经在1.7Å分辨率下确定了野生型FRasel的晶体结构。本研究以双香豆素、羟基香豆素、华法林等为研究对象，研究了该化合物的结构。经X-射线结晶学测定。由于Phe124参与了FRasel的活性，我们还产生了用Phe124替代的各种突变体。并对这些突变体进行了酶反应动力学分析。对于Phe124Ala突变体和Phe124Trp突变体，只对突变体和与抑制剂的络合物进行了X射线结晶学研究。因此，我们可以考虑表达底物特异性的残基。虽然这个含有FRasel、NFSA等的酶家族具有黄素还原活性、硝基还原活性和苯醌还原活性，但我们明确了它们是根据底物活性最高的底物进行分类的。将这些突变体用于活性筛选，获得了一些具有活性的突变体。其中，用另外11个氨基酸取代Phe42产生突变体，并对其NADPH氧化活性进行了分析。结果表明，底物连接口袋中的苯环结构有助于NADPH的特异性。

The flavin reductase of luminous bacterium has a close relation to a luminescence phenomenon, and the nitro reductase is related to mutation. In this research, we investigated both wild type and mutant of the flavin reductase (FRasel from luminous baderium Vivno fischerii) and the nitro reductase (NfsA from E Coli.) by X-ray crystallography. The purpose is to clarify mechanism of the enzyme reaction and substrate specificity on an atomic level.We have already determined the crystal structure of wild type FRasel in 1.7Å resolution. In this research, the structures of the complex with inhibitors (dicoumarol, hydroxycoumarin, warfarin, etc.) were determined by X-ray crystallography. We also produced various mutants with replaced Phe124 because Phe124 participates in the activity of FRasel. And we performed enzyme reaction kinetics analysis using those mutants. Regarding the Phe124Ala mutant and the Phe124Trp mutant, X-ray crystallography of the mutant only and the complex with the inhibitor was performed. As a result, we could consider the residue expressing substrate specificity.Although This enzyme family containing FRasel, NfsA, etc. has all of flavin reduction activity, nitro reduction activity and quinone reduction activity, we have made it clear that they are classified according to the substrate with the highest activity.Regarding the NfsA, random mutation was introduced by PCR. These mutants were applied to activity screening, and some mutants showing activity were acquired. Among these, the mutants with Phe42 replaced by another 11 amino acids were created and the NADPH oxidization activity was analyzed. Consequently, it was shown that the benzene ring structure in a substrate joint pocket contributed to the specificity of NADPH.