A structural element that facilitates proton-coupled electron transfer in oxalate decarboxylase.
A structural element that facilitates proton-coupled electron transfer in oxalate decarboxylase.
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一种促进草酸脱羧酶中质子耦合电子转移的结构元件。
DOI：
10.1021/bi300001q
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发表时间：
2012
期刊：
影响因子：
2.9
通讯作者：
Richards,NigelGJ
中科院分区：
文献类型：
--
作者：
Saylor,BenjaminT;Reinhardt,LaurieA;Lu,Zhibing;Shukla,MithilaS;Nguyen,Linda;Cleland,WWallace;Angerhofer,Alexander;Allen,KarenN;Richards,NigelGJ
由残基Ser161-Glu162-Asn163-Ser164定义的活性位点环段的构象性质已被证明对调节枯草异草酸芽孢杆菌脱羧酶活性位点Mn（II）的内在反应性很重要。我们现在详细介绍了通过位点特异性诱变去除保守的Arg/Thr氢键相互作用的功能和结构后果。因此，用缬氨酸残基取代Thr-165得到催化活性受损的OxDC变体（T165V）。重原子同位素效应测量，结合T165V型OxDC的x射线晶体结构，证明了保守的Arg/Thr氢键对于正确定位Glu-162侧链是重要的，在催化型OxDC中，Glu-162侧链介导质子耦合电子转移（PCET）步进脱羧。此外，我们还发现，T165V氧化dc变体显示出每个双氧分子的草酸消耗水平较低，这与最近的自旋捕获实验的预测一致[Imaram et al. (2011)Free radical Biol]。中华医学杂志，1999,19(2):444 - 444。这一发现表明，在两个假定的PCET步骤中，双氧可能作为可逆电子汇参与，而不仅仅是用于产生基于蛋白质的自由基或氧化的金属中心。
The conformational properties of an active-site loop segment, defined by residues Ser161-Glu162-Asn163-Ser164, have been shown to be important for modulating the intrinsic reactivity of Mn(II) in the active site ofBacillus subtilisoxalate decarboxylase. We now detail the functional and structural consequences of removing a conserved Arg/Thr hydrogen-bonding interaction by site-specific mutagenesis. Hence, substitution of Thr-165 by a valine residue gives an OxDC variant (T165V) that exhibits impaired catalytic activity. Heavy-atom isotope effect measurements, in combination with the X-ray crystal structure of the T165V OxDC variant, demonstrate that the conserved Arg/Thr hydrogen bond is important for correctly locating the side chain of Glu-162, which mediates a proton-coupled electron transfer (PCET) steppriorto decarboxylation in the catalytically competent form of OxDC. In addition, we show that the T165V OxDC variant exhibits a lower level of oxalate consumption per dioxygen molecule, consistent with the predictions of recent spin-trapping experiments [Imaram et al. (2011)Free Radicals Biol. Med. 50, 1009–1015]. This finding implies that dioxygen might participate as a reversible electron sink in two putative PCET steps and is not merely used to generate a protein-based radical or oxidized metal center.