A mutational analysis of active site residues in trans-3-chloroacrylic acid dehalogenase

• Published in: FEBS letters 2013-09, Vol.587 (17), p.2842-2850
• Main Authors: Poelarends, Gerrit J., Serrano, Hector, Huddleston, Jamison P., Johnson, William H., Whitman, Christian P.
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Catalytic Domain; Enzyme mechanism; Hydrogen Bonding; Hydrogen-Ion Concentration; Hydrolases - chemistry; Hydrolases - genetics; Hydrolytic dehalogenation; Kinetics; Molecular Docking Simulation; Molecular Weight; Mutagenesis, Site-Directed; Mutant Proteins - chemistry; Mutant Proteins - genetics; Protein Binding; Protein Structure, Secondary; Pseudomonas - enzymology; Tautomerase superfamily; trans-3-Chloroacrylic acid dehalogenase
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/j.febslet.2013.07.006

trans-3-Chloroacrylic acid dehalogenase (CaaD) catalyzes the hydrolytic dehalogenation of trans-3-haloacrylates to yield malonate semialdehyde by a mechanism utilizing βPro-1, αArg-8, αArg-11, and αGlu-52. These residues are implicated in a promiscuous hydratase activity where 2-oxo-3-pentynoate is processed to acetopyruvate. The roles of three nearby residues (βAsn-39, αPhe-39, and αPhe-50) are unexplored. Mutants were constructed at these positions (βN39A, αF39A, αF39T, αF50A and αF50Y) and kinetic parameters determined along with those of the αR8K and αR11K mutants. Analysis indicates that αArg-8, αArg-11, and βAsn-39 are critical for dehalogenase activity whereas αArg-11 and αPhe-50 are critical for hydratase activity. Docking studies suggest structural bases for these observations.