Cloning, expression, and characterization of a cis-3-chloroacrylic acid dehalogenase:: Insights into the mechanistic, structural, and evolutionary relationship between isomer-specific 3-chloroacrylic acid dehalogenases
Cloning, expression, and characterization of a cis-3-chloroacrylic acid dehalogenase:: Insights into the mechanistic, structural, and evolutionary relationship between isomer-specific 3-chloroacrylic acid dehalogenases
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DOI：
10.1021/bi0355948
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发表时间：
2004-01-27
期刊：
影响因子：
2.9
通讯作者：
Whitman, CP
中科院分区：
文献类型：
--
作者：
Poelarends, GJ;Serrano, H;Whitman, CP
从棒状杆菌FG41菌株中克隆并高效表达了顺式-3-氯丙烯酸脱卤酶(cis-CAAD)基因，对该酶进行了纯化，并对其进行了动力学和力学性质的研究。动力学研究表明，顺-CAAD处理顺式-3-卤代丙烯酸酯，而不是反式-3-卤代丙烯酸酯，其周转数接近10 S(-1)。反应产物为丙二酸半醛，其特征核磁共振氢谱证实了该产物的存在。该酶与先前研究的反式-3-氯丙烯酸脱卤酶(CAAD)以及4-草酰基互换错构酶(4-OT)家族的其他成员有较低但显著的序列相似性。4-OT和CAAD分别作为同六聚体和异六聚体发挥作用，而通过凝胶过滤层析分析，顺式-CAAD似乎是一种同源三聚体蛋白。根据已知的CAAD和4-OT的三维结构和反应机制，序列比对表明Pro-1、Arg-70、Arg-73和Glu-114是顺式CAAD中重要的活性位点残基。随后的定点突变实验证实了这些预测。乙炔化合物2-氧代-3-戊烯酸酯和3-溴和3-氯丙酸酯被顺式CAAD加工成与先前为CAAD建立的酶催化水合反应一致的产物。2-氧代-3-戊烯酸水合生成乙酰丙酮酸，而3-卤代丙酸酯则成为修饰Pro-1的不可逆抑制剂。结果表明，cis-CAAD和CAAD具有不同的一级和四级结构，表现出不同的底物专一性和催化效率，但可能具有高度保守的催化机理。这一机制可能是独立进化的，因为序列分析表明cis-CAAD不是4-OT家族成员，而是互变擦除酶超家族中第一个特征成员，可能是由4-OT样序列的独立复制造成的。在这个超家族中发现了第五个酶家族，进一步证明了可以从4-OT类序列中创建的活性和结构的多样性。
The gene encoding the cis-3-chloroacrylic acid dehalogenase (cis-CaaD) from coryneform bacterium strain FG41 has been cloned and overexpressed, and the enzyme has been purified to homogeneity and subjected to kinetic and mechanistic characterization. Kinetic studies show that cis-CaaD processes cis-3-haloacrylates, but not trans-3-haloacrylates, with a turnover number of similar to10 s(-1). The product of the reaction is malonate semialdehyde, which was confirmed by its characteristic H-1 NMR spectrum. The enzyme shares low but significant sequence similarity with the previously studied trans-3-chloroacrylic acid dehalogenase (CaaD) and with other members of the 4-oxalocrotonate tautomerase (4-OT) family. While 4-OT and CaaD function as homo- and heterohexamers, respectively, cis-CaaD appears to be a homotrimeric protein as assessed by gel filtration chromatography. On the basis of the known three-dimensional structures and reaction mechanisms of CaaD and 4-OT, a sequence alignment implicated Pro-1, Arg-70, Arg-73, and Glu-114 as important active-site residues in cis-CaaD. Subsequent site-directed mutagenesis experiments confirmed these predictions. The acetylene compounds, 2-oxo-3-pentynoate and 3-bromo- and 3-chloropropiolate, were processed by cis-CaaD to products consistent with an enzyme-catalyzed hydration reaction previously established for CaaD. Hydration of 2-oxo-3-pentynoate afforded acetopyruvate, while the 3-halopropiolates became irreversible inhibitors that modified Pro-1. The results of this work revealed that cis-CaaD and CaaD have different primary and quaternary structures, and display different substrate specificity and catalytic efficiencies, but likely share a highly conserved catalytic mechanism. The mechanism may have evolved independently because sequence analysis indicates that cis-CaaD is not a 4-OT family member, but represents the first characterized member of a new family in the tautomerase superfamily that probably resulted from an independent duplication of a 4-OT-like sequence. The discovery of a fifth family of enzymes within this superfamily further demonstrates the diversity of activities and structures that can be created from 4-OT-like sequences.