A closed conformation of Bacillus subtilis oxalate decarboxylase OxdC provides evidence for the true identity of the active site
A closed conformation of Bacillus subtilis oxalate decarboxylase OxdC provides evidence for the true identity of the active site
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DOI：
10.1074/jbc.m313820200
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发表时间：
2004-05-07
影响因子：
4.8
通讯作者：
Bornemann, S
中科院分区：
文献类型：
--
作者：
Just, VJ;Stevenson, CEM;Bornemann, S
草酸脱羧酶（EC 4.1.1.2）催化草酸转化为甲酸和二氧化碳，并利用分子氧作为辅因子。相比之下，进化上相关的草酸氧化酶（EC 1.2.3.4）将草酸盐和分子氧转化为二氧化碳和过氧化氢。不同的自由基催化机制已经提出了这些酶，涉及在脱羧酶的活性位点质子供体的要求，但不是氧化酶反应。氧化酶每个亚基只有一个结构域和锰结合位点，而脱羧酶每个亚基有两个结构域和两个锰位点。脱羧酶的结构以及有限的诱变研究最近被解释为C-末端结构域锰结合位点（位点2）是催化位点并且Glu-333是关键的质子供体的证据（Anand，R.，多雷斯坦，Kinnessy角，Begley，T. P.，和Ealick，S. E.（2002）Biochemistry 41，7659-7669）。该结构的N-末端结合位点（位点1）是溶剂暴露的（开放的），并且缺乏用于脱羧酶反应的合适的质子供体。我们报告了一个新的结构的脱羧酶，显示了一个环含有一个310螺旋附近的网站1在一个替代的构象。该环采用“闭合”构造，形成覆盖部位1的入口的盖。这种构象变化使Glu-162接近锰离子，使其成为关键质子供体的新候选者。每个结构域中等同残基的定点诱变提供了证据，证明Glu-162发挥这一重要作用，并且N-末端结构域是唯一的或占主导地位的催化活性结构域。
Oxalate decarboxylase (EC 4.1.1.2) catalyzes the conversion of oxalate to formate and carbon dioxide and utilizes dioxygen as a cofactor. By contrast, the evolutionarily related oxalate oxidase (EC 1.2.3.4) converts oxalate and dioxygen to carbon dioxide and hydrogen peroxide. Divergent free radical catalytic mechanisms have been proposed for these enzymes that involve the requirement of an active site proton donor in the decarboxylase but not the oxidase reaction. The oxidase possesses only one domain and manganese binding site per subunit, while the decarboxylase has two domains and two manganese sites per subunit. A structure of the decarboxylase together with a limited mutagenesis study has recently been interpreted as evidence that the C-terminal domain manganese binding site (site 2) is the catalytic site and that Glu-333 is the crucial proton donor (Anand, R., Dorrestein, P. C., Kinsland, C., Begley, T. P., and Ealick, S. E. (2002) Biochemistry 41, 7659-7669). The N-terminal binding site (site 1) of this structure is solvent-exposed (open) and lacks a suitable proton donor for the decarboxylase reaction. We report a new structure of the decarboxylase that shows a loop containing a 310 helix near site 1 in an alternative conformation. This loop adopts a "closed" conformation forming a lid covering the entrance to site 1. This conformational change brings Glu-162 close to the manganese ion, making it a new candidate for the crucial proton donor. Site-directed mutagenesis of equivalent residues in each domain provides evidence that Glu-162 performs this vital role and that the N-terminal domain is either the sole or the dominant catalytically active domain.