Structure-Based Mutational Studies of Substrate Inhibition of Betaine Aldehyde Dehydrogenase BetB from Staphylococcus aureus
Structure-Based Mutational Studies of Substrate Inhibition of Betaine Aldehyde Dehydrogenase BetB from Staphylococcus aureus
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DOI：
10.1128/aem.00215-14
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发表时间：
2014-07-01
影响因子：
4.4
通讯作者：
Yakunin, Alexander F.
中科院分区：
文献类型：
--
作者：
Chen, Chao;Joo, Jeong Chan;Yakunin, Alexander F.
高浓度底物和/或辅因子对酶活性的抑制是在许多酶（包括醛脱氢酶）中证明的普遍现象。在这里，我们表明，未表征的蛋白质BetB（SA 2613）从金黄色葡萄球菌是一种高度特异性的甜菜碱醛脱氢酶，其表现出底物抑制在浓度低至0.15 mM的甜菜碱醛。相反，醛脱氢酶YdcW从大肠杆菌，这也是对甜菜碱醛的活性，显示没有抑制该基板。利用BetB和YdcW的晶体结构，我们对BetB进行了基于结构的突变分析，并将YdcW残基引入BetB活性位点。在总共32个突变中，位于底物结合口袋中的5个残基（Val 288、Ser 290、His 448、Tyr 450和Trp 456）中的那些突变大大降低了BetB的底物抑制，而双突变蛋白H448 F/Y 450 L表现出完全丧失底物抑制。底物抑制作用也因BetB NAD（+）结合位点上的Gly 234突变（突变为Ser、Thr或Ala）而降低，表明辅因子和底物结合位点之间存在一定的协同性。底物对接分析的BetB和YdcW的活性位点显示，野生型BetB可以结合甜菜碱醛在生产和非生产构象，而只有生产结合模式可以建模的YdcW和BetB突变体蛋白的活性位点，减少底物抑制。因此，我们的研究结果表明，底物抑制BetB的分子机制与甜菜碱醛的非生产性结合。
Inhibition of enzyme activity by high concentrations of substrate and/or cofactor is a general phenomenon demonstrated in many enzymes, including aldehyde dehydrogenases. Here we show that the uncharacterized protein BetB (SA2613) from Staphylococcus aureus is a highly specific betaine aldehyde dehydrogenase, which exhibits substrate inhibition at concentrations of betaine aldehyde as low as 0.15 mM. In contrast, the aldehyde dehydrogenase YdcW from Escherichia coli, which is also active against betaine aldehyde, shows no inhibition by this substrate. Using the crystal structures of BetB and YdcW, we performed a structure-based mutational analysis of BetB and introduced the YdcW residues into the BetB active site. From a total of 32 mutations, those in five residues located in the substrate binding pocket (Val288, Ser290, His448, Tyr450, and Trp456) greatly reduced the substrate inhibition of BetB, whereas the double mutant protein H448F/Y450L demonstrated a complete loss of substrate inhibition. Substrate inhibition was also reduced by mutations of the semiconserved Gly234 (to Ser, Thr, or Ala) located in the BetB NAD(+) binding site, suggesting some cooperativity between the cofactor and substrate binding sites. Substrate docking analysis of the BetB and YdcW active sites revealed that the wild-type BetB can bind betaine aldehyde in both productive and nonproductive conformations, whereas only the productive binding mode can be modeled in the active sites of YdcW and the BetB mutant proteins with reduced substrate inhibition. Thus, our results suggest that the molecular mechanism of substrate inhibition of BetB is associated with the nonproductive binding of betaine aldehyde.