Oxalate decarboxylase requires manganese and dioxygen for activity -: Overexpression and characterization of Bacillus subtilis YvrK and YoaN
Oxalate decarboxylase requires manganese and dioxygen for activity -: Overexpression and characterization of Bacillus subtilis YvrK and YoaN
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DOI：
10.1074/jbc.m107202200
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发表时间：
2001-11-23
影响因子：
4.8
通讯作者：
Bornemann, S
中科院分区：
文献类型：
--
作者：
Tanner, A;Bowater, L;Bornemann, S
枯草芽孢杆菌草酸脱羧酶（EC 4.1.1.2）， YvrK，将草酸转化为甲酸和二氧化碳。来自枯草芽孢杆菌的YvrK和相关假设蛋白YoaN和YxaG在大肠杆菌中成功过表达。重组YvrK和YoaN只有在锰盐存在下表达时才具有草酸脱羧酶活性。目前还没有检测到YxaG的酶活性，它以可溶性蛋白的形式表达，不需要锰盐。YvrK和YoaN可催化少量副反应：草酸氧化生成H2O2；以及草酸依赖、h2o2不依赖的染料氧化。纯化的YvrK草酸脱羧酶活性是o -2依赖性的。YvrK被发现含有0.86到1.14个锰原子/亚基。EPR光谱显示金属离子主要处于Mn(H)氧化态，但不完全处于Mn(H)氧化态。主g = 2信号的超细耦合常数（A = 9.5 militesla）与氧、氮配体具有六坐标几何结构。YvrK的结构与草酸氧化酶、菜豆蛋白和菜豆蛋白具有同源性，并与前甘氨酸和均质1,2-双加氧酶相似，预测其六聚体低聚。虽然YvrK具有两个潜在的活性位点，但只有一个可以被锰完全占据。讨论了六聚体酶中c端结构域活性位点没有锰键而埋在亚基间界面的可能性。提出了一种草酸脱羧的机制，其中Mn（II）和O-2都是辅助因子，在催化过程中作为双电子汇一起起作用。
The Bacillus subtilis oxalate decarboxylase (EC 4.1.1.2), YvrK, converts oxalate to formate and CO2. YvrK and the related hypothetical proteins YoaN and YxaG from B. subtilis have been successfully overexpressed in Escherichia coli. Recombinant YvrK and YoaN were found to be soluble enzymes with oxalate decarboxylase activity only when expressed in the presence of manganese salts. No enzyme activity has yet been detected for YxaG, which was expressed as a soluble protein without the requirement for manganese salts. YvrK and YoaN were found to catalyze minor side reactions: oxalate oxidation to produce H2O2; and oxalate-dependent, H2O2-independent dye oxidations. The oxalate decarboxylase activity of purified YvrK was O-2-dependent. YvrK was found to contain between 0.86 and 1.14 atoms of manganese/subunit. EPR spectroscopy showed that the metal ion was predominantly but not exclusively in the Mn(H) oxidation state. The hyperfine coupling constant (A = 9.5 millitesla) of the main g = 2 signal was consistent with oxygen and nitrogen ligands with hexacoordinate geometry. The structure of YvrK was modeled on the basis of homology with oxalate oxidase, canavalin, and phaseolin, and its hexameric oligomerization was predicted by analogy with proglycinin and homogentisate 1,2-dioxygenase. Although YvrK possesses two potential active sites, only one could be fully occupied by manganese. The possibility that the C-terminal domain active site has no manganese bound and is buried in an intersubunit interface within the hexameric enzyme is discussed. A mechanism for oxalate decarboxylation is proposed, in which both Mn(II) and O-2 are cofactors that act together as a two-electron sink during catalysis.