Structure determination and functional analysis of a chromate reductase from Gluconacetobacter hansenii.
Structure determination and functional analysis of a chromate reductase from Gluconacetobacter hansenii.
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DOI：
10.1371/journal.pone.0042432
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Long PE
中科院分区：
文献类型：
--
作者：
Jin H;Zhang Y;Buchko GW;Varnum SM;Robinson H;Squier TC;Long PE
通过有助于有毒金属还原固定的生物机制进行环境保护（例如，铬酸盐和铀酰）已被鉴定涉及促进细胞活力的特异性NADH依赖性黄素蛋白。为了了解负责金属还原的酶机制，测定了来自汉逊葡糖酸醋杆菌（Gluconacetobacter hansenii）的推定铬酸盐还原酶（Gh-ChrR）的酶动力学，并在2.25 nm分辨率下测定了蛋白质的晶体结构。在有氧条件下，Gh-ChrR催化铬酸根、铁氰化物和铀酰阴离子的还原。动力学测量表明，NADH作为一种底物抑制剂，催化需要铬酸盐结合之前，NADH协会。Gh-ChrR的晶体结构表明该蛋白是一个同源四聚体，每个亚基有一个结合的黄素单核苷酸（FMN）。一个结合阴离子可视化近端的FMN在相邻的亚基之间的界面内的阳离子口袋，这是定位在一个最佳的距离氢化物转移。建议参与NADH和金属阴离子结合的残基（N85 A或R101 A）的定点取代导致NADH依赖性铬酸盐还原的酶效率降低90-95%。相比之下，参与活性位点中FMN配位的残基（S118 A）的定点取代仅导致催化效率的适度（50%）降低，这与将FMN定位在活性位点中的大量侧链的存在一致。金属阴离子结合位点和酶辅因子之间的建议接近关系进行了讨论，在合理的设计原则，使用酶在铬酸盐和铀酰生物修复。
Environmental protection through biological mechanisms that aid in the reductive immobilization of toxic metals (e.g., chromate and uranyl) has been identified to involve specific NADH-dependent flavoproteins that promote cell viability. To understand the enzyme mechanisms responsible for metal reduction, the enzyme kinetics of a putative chromate reductase from Gluconacetobacter hansenii (Gh-ChrR) was measured and the crystal structure of the protein determined at 2.25 Å resolution. Gh-ChrR catalyzes the NADH-dependent reduction of chromate, ferricyanide, and uranyl anions under aerobic conditions. Kinetic measurements indicate that NADH acts as a substrate inhibitor; catalysis requires chromate binding prior to NADH association. The crystal structure of Gh-ChrR shows the protein is a homotetramer with one bound flavin mononucleotide (FMN) per subunit. A bound anion is visualized proximal to the FMN at the interface between adjacent subunits within a cationic pocket, which is positioned at an optimal distance for hydride transfer. Site-directed substitutions of residues proposed to involve in both NADH and metal anion binding (N85A or R101A) result in 90–95% reductions in enzyme efficiencies for NADH-dependent chromate reduction. In comparison site-directed substitution of a residue (S118A) participating in the coordination of FMN in the active site results in only modest (50%) reductions in catalytic efficiencies, consistent with the presence of a multitude of side chains that position the FMN in the active site. The proposed proximity relationships between metal anion binding site and enzyme cofactors is discussed in terms of rational design principles for the use of enzymes in chromate and uranyl bioremediation.