A mutational analysis of the active site loop residues in cis-3-Chloroacrylic acid dehalogenase.
A mutational analysis of the active site loop residues in cis-3-Chloroacrylic acid dehalogenase.
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DOI：
10.1021/bi4004414
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发表时间：
2013-06-18
期刊：
影响因子：
2.9
通讯作者：
Whitman, Christian P.
中科院分区：
文献类型：
--
作者：
Schroeder, Gottfried K.;Huddleston, Jamison P.;Johnson, William H., Jr.;Whitman, Christian P.
来自 Pseudomonas pavonaceae 170 的顺式 -3-氯丙烯酸脱卤酶 (cis-CaaD) 和来自谷氨酸棒杆菌的 Cg10062 同源物具有 34% 的序列同一性（54% 相似性）。前者催化杀线虫剂 1,3-二氯丙烯的细菌分解代谢途径中的关键步骤，而后者没有已知的生物活性。尽管 Cg10062 具有对 cis-CaaD 活性至关重要的 6 个活性位点残基（Pro-1、His-28、Arg-70、Arg-73、Tyr-103、Glu-114），但它仅表现出低水平的 cis-CaaD 活性并且缺乏 cis-CaaD 的特异性：Cg10062 处理 3-氯丙烯酸酯的两种异构体，并优先处理顺式异构体。尽管这些差异的基础尚不清楚，但通过与相同抑制剂孵育产生的加合物共价修饰的酶的晶体结构的比较提供了可能的解释。 cis-CaaD 中的 6 残基活性位点环显示出与 Cg10062 中观察到的显着不同的构象：该环在 cis-CaaD 的活性位点上闭合，但在 Cg10062 的活性位点上闭合。为了检查该环对 cis-CaaD 催化和特异性的贡献，将残基单独更改为 Cg10062 中发现的残基。随后的动力学和机制分析表明 cis-CaaD 的 T34A 突变体更像 Cg10062。突变酶显示 Km 增加了 4 倍（使用顺式 3-溴丙烯酸酯），但未达到 Cg10062 观察到的程度（687 倍）。该突变还导致突发率降低 4 倍（与野生型 cis-CaaD 相比），而 Cg10062 没有显示突发率。更能说明问题的是 cis-CaaD 的 T34A 突变体与替代底物 2,3-丁二烯酸的反应。在 NaBH4 和丙二烯存在的情况下，由于 Pro-1 的共价修饰，cis-CaaD 在一次周转后完全失活。使用 Cg10062 进行的相同实验未导致 Pro-1 的共价修饰。不同的结果归因于共价催化（使用 Pro-1），随后顺式 CaaD 中的烯胺或亚胺互变异构体水解，而在 Cg10062 的情况下，丙二烯直接水合产生乙酰乙酸。 T34A 突变体表现出部分失活，每个单体需要 5 次底物周转，这表明 80% 的时间倾向于直接水合途径。然而，当反应在 D2O 中进行时，突变不会改变 [2-D]乙酰乙酸酯 C-2 的立体化学。 cis-CaaD 和 T34 突变体均生成 (2R)-[2-D]乙酰乙酸，而 Cg10062 主要生成 2S 异构体。综合观察结果与环区在 cis-CaaD 特异性和催化中的作用一致，但确切的作用仍有待确定。
cis -3-Chloroacrylic acid dehalogenase (cis-CaaD) from Pseudomonas pavonaceae 170 and a homologue from Corynebacterium glutamicum designated Cg10062 share 34% sequence identity (54% similarity). The former catalyzes a key step in a bacterial catabolic pathway for the nematocide 1,3-dichloropropene, whereas the latter has no known biological activity. Although Cg10062 has the six active site residues (Pro-1, His-28, Arg-70, Arg-73, Tyr-103, Glu-114) that are critical for cis-CaaD activity, it shows only a low level cis-CaaD activity and lacks the specificity of cis-CaaD: Cg10062 processes both isomers of 3-chloroacrylate with a preference for the cis-isomer. Although the basis for these differences is unknown, a comparison of the crystal structures of the enzymes covalently modified by an adduct resulting from their incubation with the same inhibitor offers a possible explanation. A 6-residue active site loop in cis-CaaD shows a strikingly different conformation from that observed in Cg10062: the loop closes down on the active site of cis-CaaD, but not on that of Cg10062. In order to examine what this loop might contribute to cis-CaaD catalysis and specificity, the residues were changed individually to those found in Cg10062. Subsequent kinetic and mechanistic analysis suggests that the T34A mutant of cis-CaaD is more Cg10062-like. The mutant enzyme shows a 4-fold increase in Km (using cis-3-bromoacrylate), but not to the degree observed for Cg10062 (687-fold). The mutation also causes a 4-fold decrease in the burst rate (compared to the wild type cis-CaaD), whereas Cg10062 shows no burst rate. More telling is the reaction of the T34A mutant of cis-CaaD with the alternate substrate, 2,3-butadienoate. In the presence of NaBH4 and the allene, cis-CaaD is completely inactivated after one turnover due to the covalent modification of Pro-1. The same experiment with Cg10062 does not result in the covalent modification of Pro-1. The different outcomes are attributed to covalent catalysis (using Pro-1) followed by hydrolysis of the enamine or imine tautomer in cis-CaaD vs direct hydration of the allene to yield acetoacetate in the case of Cg10062. The T34A mutant shows partial inactivation, requiring 5 turnovers of the substrate per monomer, which suggests that the direct hydration route is favored 80% of the time. However, the mutation does not alter the stereochemistry at C-2 of [2-D]acetoacetate when the reaction is carried out in D2O. Both cis-CaaD and the T34 mutant generate (2R)-[2-D]acetoacetate, whereas Cg10062 generates mostly the 2S-isomer. The combined observations are consistent with a role for the loop region in cis-CaaD specificity and catalysis, but the precise role remains to be determined.
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DOI：
10.1099/00221287-137-8-2025
发表时间：
1991-08-01
期刊：
JOURNAL OF GENERAL MICROBIOLOGY
影响因子：
--
作者：
HARTMANS, S;JANSEN, MW;DEBONT, JAM
通讯作者：
DEBONT, JAM
影响因子：
2.9
作者：
Poelarends, GJ;Serrano, H;Whitman, CP
通讯作者：
Whitman, CP
影响因子：
2.9
作者：
Johnson, Kenneth A.;Simpson, Zachary B.;Blom, Thomas
通讯作者：
Blom, Thomas
影响因子：
4.8
作者：
de Jong, RM;Brugman, W;Dijkstra, BW
通讯作者：
Dijkstra, BW
影响因子：
2.9
作者：
Nguyen, Henry H.;Wang, Liangbing;Huang, Hua;Peisach, Ezra;Dunaway-Mariano, Debra;Allen, Karen N.
通讯作者：
Allen, Karen N.