Crystal structures of native and inactivated cis-3-chloroacrylic acid dehalogenase. Structural basis for substrate specificity and inactivation by (R)-oxirane-2-carboxylate.
The bacterial degradation pathways for the nematocide 1,3-dichloropropene rely on hydrolytic dehalogenation reactions catalyzed by cis- and trans-3-chloroacrylic acid dehalogenases (cis-CaaD and CaaD, respectively). X-ray crystal structures of native cis-CaaD and cis-CaaD inactivated by (R)-oxirane-2-carboxylate were elucidated. They locate four known catalytic residues (Pro-1, Arg-70, Arg-73, and Glu-114) and two previously unknown, potential catalytic residues (His-28 and Tyr-103'). The Y103F and H28A mutants of these latter two residues displayed reductions in cis-CaaD activity confirming their importance in catalysis. The structure of the inactivated enzyme shows covalent modification of the Pro-1 nitrogen atom by (R)-2-hydroxypropanoate at the C3 position. The interactions in the complex implicate Arg-70 or a water molecule bound to Arg-70 as the proton donor for the epoxide ring-opening reaction and Arg-73 and His-28 as primary binding contacts for the carboxylate group. This proposed binding mode places the (R)-enantiomer, but not the (S)-enantiomer, in position to covalently modify Pro-1. The absence of His-28 (or an equivalent) in CaaD could account for the fact that CaaD is not inactivated by either enantiomer. The cis-CaaD structures support a mechanism in which Glu-114 and Tyr-103' activate a water molecule for addition to C3 of the substrate and His-28, Arg-70, and Arg-73 interact with the C1 carboxylate group to assist in substrate binding and polarization. Pro-1 provides a proton at C2. The involvement of His-28 and Tyr-103' distinguishes the cis-CaaD mechanism from the otherwise parallel CaaD mechanism. The two mechanisms probably evolved independently as the result of an early gene duplication of a common ancestor.
de Jong
René M
RM
Laboratory of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
Bazzacco
Paola
P
Poelarends
Gerrit J
GJ
Johnson
William H
WH
Kim
Yoon Jae
YJ
Burks
Elizabeth A
EA
Serrano
Hector
H
Thunnissen
Andy-Mark W H
AM
Whitman
Christian P
CP
Dijkstra
Bauke W
BW
D000595:Amino Acid Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D004852:Epoxy Compounds / Q000737:chemistry / Q000378:metabolism; D005027:Ethylene Oxide / Q000737:chemistry / Q000378:metabolism; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011549:Pseudomonas; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D004852:Epoxy Compounds; D006867:Hydrolases; C077719:cis-3-chloroacrylic acid dehalogenase; D005027:Ethylene Oxide