Mechanistic and structural studies of bacterial catabolic enzymes : trans-and cis-3-chloroacrylic acid dehalogenases and YegM
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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). Based on X-ray crystal structures of native cis-CaaD and cis-CaaD inactivated by (R)-oxirane-2-carboxylate, the roles of four known catalytic residues (Pro-1, Arg-70, Arg-73, and Glu-114) have been studied further and two previously unknown, potential catalytic residues (His-28 and Tyr-103) were identified. 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 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 roles of Tyr-103 and His-28 in the mechanism and inhibition of cis-CaaD have been studied. An E. coli enzyme designated YcgM is a member of the fumarylacetoacetate hydrolase (FAH) family in a FAH superfamily. Little is known about YcgM from E. coli C, but the structure is reported in the data base. Based on a structural comparison with the Cterminal part of 5-carboxymethyl-2-oxo-3-hexene-1,6-dioate decarboxylase (COHED), YcgM does not have a catalytic dyad, but may have an oxyanion hole. YcgM has been cloned expressed, and purified. 2-hydroxy-2,4-pentadienoate (HPD) was found to undergo an enzymatic reaction by YcgM. However, the significance of this enzymatic reaction rate is unknown. With the newly found substrate, a divalent metal and pH dependence of YcgM has been examined.