X-Ray Crystallography of Flavin Reductase and Its Mutants

发光菌的黄素还原酶与发光现象密切相关，硝基还原酶与突变有关。在本研究中，我们研究了野生型和突变型的黄素还原酶（FRasel从发光Baderium Vivno fischerii）和硝基还原酶（NfsA从大肠杆菌）。通过X射线晶体学。目的是在原子水平上阐明酶的反应机理和底物特异性。我们已经确定了野生型FRasel的晶体结构，分辨率为1.7 nm。本文研究了抑制剂（双香豆素、羟基香豆素、华法林等）的配合物的结构。通过X射线晶体学测定。由于Phe124参与FRasel的活性，我们还产生了各种替换Phe124的突变体。并利用这些突变体进行了酶反应动力学分析。关于Phe124Ala突变体和Phe124Trp突变体，仅对突变体和与抑制剂的复合物进行X射线晶体学分析。结果，我们可以考虑表达底物特异性的残基。虽然包含FRasel、NfsA等的该酶家族具有所有的黄素还原活性、硝基还原活性和醌还原活性，但我们已经明确它们是根据具有最高活性的底物分类的。关于NfsA，通过PCR引入随机突变。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，Phe42被另外11个氨基酸取代的突变体被创建，并分析NADPH氧化活性。结果表明，底物结合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.