Specificity versus catalytic potency: The role of threonine 44 in Escherichia coli dihydrodipicolinate synthase mediated catalysis
Specificity versus catalytic potency: The role of threonine 44 in Escherichia coli dihydrodipicolinate synthase mediated catalysis
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DOI：
10.1016/j.biochi.2009.05.013
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发表时间：
2009-08-01
期刊：
影响因子：
3.9
通讯作者：
Gerrard, Juliet A.
中科院分区：
文献类型：
--
作者：
Dobson, Renwick C. J.;Perugini, Matthew A.;Gerrard, Juliet A.
在植物和细菌中，（S）-赖氨酸生物合成的分支点是（S）-酒石酸-β-半醛和丙酮酸的缩合，这是由二氢吡啶二羧酸合酶（DHDPS，E.C. 4.2.1.52）。在这项研究中，我们探测苏氨酸44在大肠杆菌DHDPS的功能，相对于其在质子中继的作用。通过突变为缬氨酸去除苏氨酸44的羟基部分，显著减弱活性（野生型的0.1%），因为质子中继被破坏。因此，预测苏氨酸44突变为丝氨酸将重新建立质子中继，从而重新建立酶活性。在定点诱变和纯化以产生DHDPS-Thr 44 Ser突变酶之后，进行动力学和结构研究。DHDPS-Thr 44 Ser的晶体结构显示活性位点是完整的，并且Ser 44和Tyr 107具有一定的构象柔性，这与观察到的与野生型酶相比的活性损失一致。在DHDPS-Thr 44 Ser的活性位点处观察到电子密度，其被鉴定为捕获的丙酮酸类似物α-酮戊二酸。确实发现活性相对于DHDPS-Thr 44 Val增加，但仍然降低至仅与野生型酶的活性的8%相似。有趣的是，有一个转变的动力学机制，从取代酶的机制，在野生型中观察到的，三元复合物的机制，与被困的底物类似物。活性位点的灵活性增加似乎有利于底物类似物的结合/反应，这表明野生型DHDPS已经进化出相对刚性的活性位点，以保持丙酮酸的底物特异性。(C)2009年Elsevier Masson SAS。All rights reserved.
In plants and bacteria, the branch point of (S)-lysine biosynthesis is the condensation of (S)-aspartate-beta-semialdehyde and pyruvate, a reaction catalysed by dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52). In this study, we probe the function of threonine 44 in Escherichia coli DHDPS, with respect to its role in the proton relay. Removal of the hydroxyl moiety of threonine 44, by mutation to valine, significantly attenuates activity (0.1% of wild-type) because the proton relay is broken. It was thus predicted that mutation of threonine 44 to serine would re-establish the proton relay and thus enzymatic activity. Following site-directed mutagenesis and purification to yield the DHDPS-Thr44Ser mutant enzyme, kinetic and structural studies were undertaken. The crystal structure of DHDPS-Thr44Ser showed that the active site was intact and that Ser44 and Tyr107 have some conformational flexibility, which is consistent with the observed loss of activity compared to the wild-type enzyme. Electron density was observed at the active site of DHDPS-Thr44Ser, which was identified as a trapped pyruvate analogue, alpha-ketoglutarate. The activity was indeed found to be increased relative to DHDPS-Thr44Val, but was still reduced to only similar to 8% of that of the wild-type enzyme. Interestingly, there was a shift in the kinetic mechanism, from the substituted-enzyme mechanism, observed in the wild-type, to the ternary-complex mechanism, consistent with the trapped substrate analogue. Increased flexibility in the active site appears to facilitate the binding/reaction of substrate analogues, suggesting that wild-type DHDPS has evolved a relatively rigid active site in order to maintain substrate specificity for pyruvate. (C) 2009 Elsevier Masson SAS. All rights reserved.