Metal Dependence of Oxalate Decarboxylase Activity
Metal Dependence of Oxalate Decarboxylase Activity
复制标题
DOI：
10.1021/bi801856k
复制
发表时间：
2009-07-07
期刊：
影响因子：
2.9
通讯作者：
Richards, Nigel G. J.
中科院分区：
文献类型：
--
作者：
Moomaw, Ellen W.;Angerhofer, Alexander;Richards, Nigel G. J.
枯草芽孢杆菌草酸脱羧酶（OxDC）催化草酸转化为CO和甲酸。该酶由两个cupin结构域组成，每个结构域都含有Mn（II）离子。虽然普遍认为Mn（II）在N-末端结构域介导OxDC催化的脱羧，但关于Mn（II）在C-末端cupin结构域中结合的功能（如果有的话）已经提出了合理的问题。我们已经研究了这个问题，使用一系列的OxDC突变体，其中Mn（II）的结合受到干扰的Glu-101和Glu-280，这协调的金属在N-末端和C-末端结构域，分别诱变。我们现在证明，脱羧酶活性和总锰含量是敏感的金属结合谷氨酸残基的修改。这些发现，结合EPR测量，提高了C-末端Mn（II）中心可以催化脱羧反应的可能性。已经从用于制备野生型OxDC的体内和体外策略的组合提供了对该结论的进一步支持，其中Mn（II）以各种程度并入。这些变体的动力学表征表明，OxDC活性与锰含量呈线性相关，如果这两个位点都能催化草酸盐分解为甲酸盐和CO2，这可能是预期的。这些研究也代表了第一个明确的证明，OxDC活性是唯一介导的锰。
Bacillus subtilis oxalate decarboxylase (OxDC) catalyzes the conversion of oxalate into CO, and formate. The enzyme is composed of two cupin domains, each of which contains a Mn(II) ion. Although there is general agreement that Mn(II) in the N-terminal domain mediates OxDC-catalyzed decarboxylation, legitimate questions have been raised concerning the function (if any) of the Mn(II) bound in the C-terminal cupin domain. We have investigated this problem using a series of OxDC mutants in which Mn(II) binding is perturbed by mutagenesis of Glu-101 and Glu-280, which coordinate the metal in the N-terminal and C-terminal domains, respectively. We now demonstrate that decarboxylase activity and total manganese content are sensitive to modifications in either metal-binding glutamate residue. These Findings, in combination with EPR measurements, raise the possibility that the C-terminal Mn(II) center can catalyze the decarboxylation reaction. Further support for this conclusion has been provided from a combination of in vivo and in vitro strategies for preparing wild-type OxDC in which Mn(II) is incorporated to a variety of extents. Kinetic characterization of these variants shows that OxDC activity is linearly correlated with manganese content, as might be expected if both sites can catalyze the breakdown of oxalate into formate and CO2. These studies also represent the first unequivocal demonstration that OxDC activity is uniquely mediated by manganese.