Abstract
D-2-haloacid dehalogenases (D-DEXs) catalyse the hydrolytic dehalogenation of D-2-haloacids, releasing halide ions and producing the corresponding 2-hydroxyacids. A structure-guided elucidation of the catalytic mechanism of this dehalogenation reaction has not been reported yet. Here, we report the catalytic mechanism of a D-DEX, HadD AJ1 from Pseudomonas putida AJ1/23, which was elucidated by X-ray crystallographic analysis and the H218O incorporation experiment. HadD AJ1 is an α-helical hydrolase that forms a homotetramer with its monomer including two structurally axisymmetric repeats. The product-bound complex structure was trapped with L-lactic acid in the active site, which is framed by the structurally related helices between two repeats. Site-directed mutagenesis confirmed the importance of the residues lining the binding pocket in stabilizing the enzyme-substrate complex. Asp205 acts as a key catalytic residue and is responsible for activating a water molecule along with Asn131. Then, the hydroxyl group of the water molecule directly attacks the C2 atom of the substrate to release the halogen ion instead of forming an enzyme-substrate ester intermediate as observed in L-2-haloacid dehalogenases. The newly revealed structural and mechanistic information on D-DEX may inspire structure-based mutagenesis to engineer highly efficient haloacid dehalogenases.
Highlights
Dehalogenases are capable of degrading a wide range of halogenated compounds by cleaving the C-X bond
More than 90% of dehalogenases cleave C-X bonds. Such C-X bonds mainly exist in the halocarbons, halohydrins, and haloacids and their derivatives8–10. 2-Haloacid dehalogenases (2-HADs) catalyse the hydrolytic dehalogenation of 2-haloacids, releasing halogen ions and producing corresponding 2-hydroxyacids. 2-HADs are phylogenetically classified into two groups, I and II11
D-DEXs act on D-2-haloacids, whereas DL-DEXs act on both D- and L-2-haloacids
Summary
Dehalogenases are capable of degrading a wide range of halogenated compounds by cleaving the C-X bond. Such enzymes are fascinating for their valuable applications in green chemistry, biocatalysis and bioremediation[1,2,3,4,5,6,7]. Group II enzymes include L-2-haloacid dehalogenases (L-DEXs) which act on L-2-haloacids. L-DEX catalyses the hydrolytic dehalogenation through an enzyme-substrate (E-S) intermediate[13]. For D-DEX, a catalytic mechanism was inferred from sequence similarity to DL-DEX It was investigated by MD simulations[1,16,17], but a structure-guided study is unavailable. The structure-guided elucidation of the catalytic mechanism of D-DEX is presented here
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