Expansion of substrate specificity and catalytic mechanism of azoreductase by X-ray crystallography and site-directed mutagenesis
Expansion of substrate specificity and catalytic mechanism of azoreductase by X-ray crystallography and site-directed mutagenesis
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DOI：
10.1074/jbc.m710070200
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发表时间：
2008-05-16
影响因子：
4.8
通讯作者：
Tanokura, Masaru
中科院分区：
文献类型：
--
作者：
Ito, Kosuke;Nakanishi, Masayuki;Tanokura, Masaru
AzoR是从大肠杆菌中分离出来的一种依赖于fmn的nadh -偶氮还原酶，是一种负责偶氮化合物降解的蛋白质。我们以前报道过这种酶在氧化状态下的晶体结构。本研究在不同条件下测定了AzoR的不同结构，以获得该酶反应机理的线索。还原后的AzoR显示出FMN辅因子的异alloxazine环的扭曲蝴蝶弯曲和溶剂分子的重排。测定了氧化后的AzoR在不同空间基上的晶体结构，以及与抑制剂双酚醇配合物的酶的结构。这些结构表明，在异alloxazine环周围形成疏水部分对底物结合很重要，Arg-59与偶氮化合物羧基之间的静电相互作用导致底物对甲基红的偏好高于对甲基红。Ala取代Arg-59使对甲基红的V-max值提高了27倍，K-m值提高了3.8倍。这表明Arg-59决定了AzoR的底物特异性。R59A突变体对甲基红还原的V-max值与野生型酶对甲基红还原的V-max值相当，但对甲基红的活性保持不变。这些发现表明AzoR底物特异性通过单个氨基酸取代而扩大。在此基础上，建立了azor -甲基红配合物的真实模型。
AzoR is an FMN-dependent NADH-azoreductase isolated from Escherichia coli as a protein responsible for the degradation of azo compounds. We previously reported the crystal structure of the enzyme in the oxidized form. In the present study, different structures of AzoR were determined under several conditions to obtain clues to the reaction mechanism of the enzyme. AzoR in its reduced form revealed a twisted butterfly bend of the isoalloxazine ring of the FMN cofactor and a rearrangement of solvent molecules. The crystal structure of oxidized AzoR in a different space group and the structure of the enzyme in complex with the inhibitor dicoumarol were also determined. These structures indicate that the formation of a hydrophobic part around the isoalloxazine ring is important for substrate binding and an electrostatic interaction between Arg-59 and the carboxyl group of the azo compound causes a substrate preference for methyl red over p-methyl red. The substitution of Arg-59 with Ala enhanced the V-max value for p-methyl red 27-fold with a 3.8-fold increase of the K-m value. This result indicates that Arg-59 decides the substrate specificity of AzoR. The V-max value for the p-methyl red reduction of the R59A mutant is comparable with that for the methyl red reduction of the wild-type enzyme, whereas the activity toward methyl red was retained. These findings indicate the expansion of AzoR substrate specificity by a single amino acid substitution. Furthermore, we built an authentic model of the AzoR-methyl red complex based on the results of the study.