Reactions of trans-3-chloroacrylic acid dehalogenase with acetylene substrates: consequences of and evidence for a hydration reaction.
Various soil bacteria use 1,3-dichloropropene, a component of the commercially available fumigants Shell D-D and Telone II, as a sole source of carbon and energy. One enzyme involved in the catabolism of 1,3-dichloropropene is trans-3-chloroacrylic acid dehalogenase (CaaD), which converts the trans-isomers of 3-bromo- and 3-chloroacrylate to malonate semialdehyde. Sequence analysis suggested a relationship between the heterohexameric CaaD and the bacterial isomerase, 4-oxalocrotonate tautomerase (4-OT), thereby distinguishing CaaD from a number of dehalogenases whose mechanisms proceed through an alkyl- or aryl-enzyme intermediate. In this study, the genes for the alpha- and beta-subunits of CaaD have been synthesized using a polymerase chain reaction-based strategy, cloned into separate plasmids, and the proteins expressed and purified as the functional heterohexamer. Subsequently, the product of the reaction was confirmed to be malonate semialdehyde by (1)H and (13)C NMR spectroscopy, and kinetic constants were determined using a UV spectrophotometric assay. In view of the proposed hydrolytic nature of the CaaD-catalyzed reaction, three acetylene compounds were investigated as substrates for the enzyme. One compound, 2-oxo-3-pentynoate, a potent active site-directed irreversible inhibitor of 4-OT, is a substrate for CaaD, and was processed to acetopyruvate with kinetic constants similar to those determined for the trans-isomers of 3-bromo- and 3-chloroacrylate. The remaining two compounds, 3-bromo- and 3-chloropropiolic acid, were transformed into potent irreversible inhibitors of CaaD. The inactivation observed for 3-bromopropiolic acid is due to the covalent modification of Pro-1 of the beta-subunit. The results provide evidence for a hydratase activity and set the stage to use the 3-halopropiolic acids as ligands in inactivated CaaD complexes that can be studied by X-ray crystallography.
Wang
Susan C
SC
Division of Medicinal Chemistry, College of Pharmacy, The University of Texas, Austin, Texas 78712-1074, USA.
Person
Maria D
MD
Johnson
William H
WH
Whitman
Christian P
CP
D000114:Acetylene / Q000737:chemistry*; D000447:Aldehydes / Q000737:chemistry; D001965:Bromides / Q000737:chemistry; D002244:Carbon / Q000737:chemistry; D018360:Crystallography, X-Ray; D006867:Hydrolases / Q000737:chemistry*; D007535:Isomerases / Q000737:chemistry; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008314:Malonates / Q000737:chemistry; D013058:Mass Spectrometry; D008956:Models, Chemical; D010957:Plasmids / Q000378:metabolism; D011422:Propionates / Q000737:chemistry; D011549:Pseudomonas / Q000201:enzymology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013053:Spectrophotometry; D014357:Trypsin / Q000494:pharmacology; D014466:Ultraviolet Rays
D000447:Aldehydes; D001965:Bromides; D008314:Malonates; D011422:Propionates; D002244:Carbon; C030290:malonic acid; D006867:Hydrolases; D014357:Trypsin; C077721:trans-3-chloroacrylic acid dehalogenase; C021918:4-oxalocrotonate tautomerase; D007535:Isomerases; D000114:Acetylene; C007022:3-chloropropionic acid; C035776:3-bromopropionic acid