The X-ray structure of trans-3-chloroacrylic acid dehalogenase reveals a novel hydration mechanism in the tautomerase superfamily
The X-ray structure of trans-3-chloroacrylic acid dehalogenase reveals a novel hydration mechanism in the tautomerase superfamily
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DOI：
10.1074/jbc.m311966200
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发表时间：
2004-03-19
影响因子：
4.8
通讯作者：
Dijkstra, BW
中科院分区：
文献类型：
--
作者：
de Jong, RM;Brugman, W;Dijkstra, BW
异构体特异性3-氯丙烯酸脱卤酶在细菌降解1,3-二氯丙烯中起作用，这种化合物在农业中用于杀死植物寄生线虫。经3-溴丙酸灭活的假单胞菌pavonaceae 170的异六聚反式-3-氯丙烯酸脱卤酶（CaaD）的晶体结构表明，α -亚基上的Glu-52被定位为3-氯丙烯酸酯和3-溴丙酸酯的C-3加成羟基的水活化碱，而β -亚基上邻近的Pro-1被定位为为C-2提供质子。两个精氨酸残基α arg -8和α arg -11与C-1羧酸基相互作用，从而使α、β不饱和酸极化。与3-氯丙烯酸酯反应生成一种不稳定的卤代丙烷-3-氯-3-羟基丙酸酯，其分解产物为丙二酸酯半醛和盐酸。然而，在失活机制中，产生丙二醇溴，使betaPro-1不可逆地烷基化。CaaD与4-草草酸互变异构酶有关，与它们共享一个n端脯氨酸。然而，在4-草草酸互变异构酶中，Pro-1作为碱参与疏水活性位点内的质子转移，而在CaaD中，酸性脯氨酸稳定在亲水活性位点。因此，CaaD活性位点环境的改变促进了在变异体酶超家族中以前未知的反应，即反式3-氯丙烯酸酯和3-溴丙酸酯的α、β不饱和键的水化。这些水合反应的机制代表了一种新的催化策略，导致碳卤素键断裂。
Isomer-specific 3-chloroacrylic acid dehalogenases function in the bacterial degradation of 1,3-dichloropropene, a compound used in agriculture to kill plant-parasitic nematodes. The crystal structure of the heterohexameric trans-3-chloroacrylic acid dehalogenase (CaaD) from Pseudomonas pavonaceae 170 inactivated by 3-bromopropiolate shows that Glu-52 in the alpha-subunit is positioned to function as the water-activating base for the addition of a hydroxyl group to C-3 of 3-chloroacrylate and 3-bromopropiolate, whereas the nearby Pro-1 in the beta-subunit is positioned to provide a proton to C-2. Two arginine residues, alphaArg-8 and alphaArg-11, interact with the C-1 carboxylate groups, thereby polarizing the alpha,beta-unsaturated acids. The reaction with 3-chloroacrylate results in the production of an unstable halohydrin, 3-chloro-3-hydroxypropanoate, which decomposes into the products malonate semialdehyde and HCl. In the inactivation mechanism, however, malonyl bromide is produced, which irreversibly alkylates the betaPro-1. CaaD is related to 4-oxalocrotonate tautomerase, with which it shares an N-terminal proline. However, in 4-oxalocrotonate tautomerase, Pro-1 functions as a base participating in proton transfer within a hydrophobic active site, whereas in CaaD, the acidic proline is stabilized in a hydrophilic active site. The altered active site environment of CaaD thus facilitates a previously unknown reaction in the tautomerase superfamily, the hydration of the alpha,beta-unsaturated bonds of trans-3-chloroacrylate and 3-bromopropiolate. The mechanism for these hydration reactions represents a novel catalytic strategy that results in carbon-halogen bond cleavage.