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

trans-3-Chloroacrylic acid dehalogenase (CaaD) catalyzes the hydrolytic dehalogenation of trans-3-haloacrylates to yield malonate semialdehyde by a mechanism utilizing beta Pro-1, alpha Arg-8, alpha Arg-11, and alpha Glu-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 (beta Asn-39, alpha Phe-39, and alpha Phe-50) are unexplored. Mutants were constructed at these positions (beta N39A, alpha F39A, alpha F39T, alpha F50A and alpha F50Y) and kinetic parameters determined along with those of the alpha R8K and alpha R11K mutants. Analysis indicates that alpha Arg-8, alpha Arg-11, and beta Asn-39 are critical for dehalogenase activity whereas alpha Arg11 and alpha Phe-50 are critical for hydratase activity. Docking studies suggest structural bases for these observations. (C) 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.