title: 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.
abstract: 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.
authors.affiliation: Division of Medicinal Chemistry, College of Pharmacy, The University of Texas, Austin, Texas 78712-1074, USA.
authors.forename: Gerrit J
authors.initials: GJ
authors.lastname: Poelarends
authors.forename: Hector
authors.initials: H
authors.lastname: Serrano
authors.forename: Maria D
authors.initials: MD
authors.lastname: Person
authors.forename: William H
authors.initials: WH
authors.lastname: Johnson
authors.forename: Alexey G
authors.initials: AG
authors.lastname: Murzin
authors.forename: Christian P
authors.initials: CP
authors.lastname: Whitman
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D003352:Corynebacterium / Q000201:enzymology / Q000235:genetics; D004791:Enzyme Inhibitors / Q000737:chemistry; D019143:Evolution, Molecular; D018698:Glutamic Acid / Q000235:genetics; D006867:Hydrolases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D007535:Isomerases / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D011392:Proline / Q000235:genetics; D017433:Protein Structure, Secondary; D011522:Protons; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D011522:Protons; D018698:Glutamic Acid; D001120:Arginine; D011392:Proline; D006867:Hydrolases; C077719:cis-3-chloroacrylic acid dehalogenase; C021918:4-oxalocrotonate tautomerase; D007535:Isomerases