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

Gerrit J Poelarends, Hector Serrano, Maria D Person, William H Johnson, Alexey G Murzin, Christian P Whitman

Research output: Contribution to journalArticleAcademicpeer-review

38 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)759-772
Number of pages14
Issue number3
Publication statusPublished - 27-Jan-2004


  • Amino Acid Sequence
  • Arginine
  • Cloning, Molecular
  • Corynebacterium
  • Enzyme Inhibitors
  • Evolution, Molecular
  • Glutamic Acid
  • Hydrolases
  • Isomerases
  • Kinetics
  • Molecular Sequence Data
  • Multigene Family
  • Mutagenesis, Site-Directed
  • Nuclear Magnetic Resonance, Biomolecular
  • Peptide Fragments
  • Proline
  • Protein Structure, Secondary
  • Protons
  • Sequence Homology, Amino Acid
  • Spectrometry, Mass, Electrospray Ionization
  • Substrate Specificity

Cite this