Poelarends Gerrit J, Serrano Hector, Person Maria D, Johnson William H, Murzin Alexey G, Whitman Christian P
Division of Medicinal Chemistry, College of Pharmacy, The University of Texas, Austin, Texas 78712-1074, USA.
Biochemistry. 2004 Jan 27;43(3):759-72. doi: 10.1021/bi0355948.
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 approximately 10 s(-1). The product of the reaction is malonate semialdehyde, which was confirmed by its characteristic 1H 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.
已克隆并过量表达了来自棒状杆菌菌株FG41的顺式-3-氯丙烯酸脱卤酶（cis-CaaD）基因，该酶已被纯化至同质，并进行了动力学和作用机制表征。动力学研究表明，cis-CaaD作用于顺式-3-卤代丙烯酸酯，而不作用于反式-3-卤代丙烯酸酯，周转数约为10 s(-1)。反应产物为丙二酸半醛，通过其特征性的1H NMR光谱得以证实。该酶与先前研究的反式-3-氯丙烯酸脱卤酶（CaaD）以及4-草酰巴豆酸互变异构酶（4-OT）家族的其他成员具有较低但显著的序列相似性。虽然4-OT和CaaD分别以同六聚体和异六聚体形式发挥作用，但通过凝胶过滤色谱评估，cis-CaaD似乎是一种同三聚体蛋白。基于CaaD和4-OT已知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cis-CaaD中重要的活性位点残基。随后的定点诱变实验证实了这些预测。乙炔化合物2-氧代-3-戊炔酸酯以及3-溴和3-氯丙炔酸酯被cis-CaaD加工成与先前为CaaD确定的酶催化水合反应一致的产物。2-氧代-3-戊炔酸酯水合生成乙酰丙酮酸，而3-卤代丙炔酸酯成为修饰Pro-1的不可逆抑制剂。这项工作的结果表明，cis-CaaD和CaaD具有不同的一级和四级结构，表现出不同的底物特异性和催化效率，但可能共享高度保守的催化机制。该机制可能是独立进化而来的，因为序列分析表明cis-CaaD不是4-OT家族成员，而是互变异构酶超家族中一个新家族的首个特征成员，这个新家族可能源于4-OT样序列的独立复制。在这个超家族中发现第五个酶家族进一步证明了由4-OT样序列产生的活性和结构的多样性。
