Insights into the molecular mechanism of dehalogenation catalyzed by D-2-haloacid dehalogenase from crystal structures

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.

Stereoselective catalysis controlled by a native leucine or variant isoleucine wing‐gatekeeper in 2‐haloacid dehalogenase

“…The catalytically crucial water at the proper position directly affects the activity of the hydrolases . Considering the catalytic efficiency of HadD AJ1, the water molecule activated by the catalytic dyad was shown to have the highest nucleophilicity at a distance of 3.0 Å in the E‐S complex and the previously reported E‐P complex . Moreover, the L288I mutant showed decreased activity to L‐2‐CPA due to that in the L288I variant bound with L‐2‐CPA, L‐2‐CPA deviates from the activated water molecule compared to D‐2‐CPA.…”

“…Group I enzymes contain DL‐2‐haloacid dehalogenases (DL‐DEX), which take both enantiomers as substrates, while D‐2‐haloacid dehalogenases (D‐DEX) only act on D‐enantiomers . Previously, based on the complex structure of a D‐DEX HadD AJ1 with its product [L‐lactic acid (L‐LA)], we reported that the D‐DEX follows the same catalytic mechanism as DL‐DEXs, which is mediated by a water molecule activated by the D205‐N131 dyad . Consequently, the high structural similarity of the D‐DEXs and DL‐DEXs creates it a pair of representative models to explain the exact stereoselective principles.…”

Genomic Analysis of Pseudomonas sp. Strain SCT, an Iodate-Reducing Bacterium Isolated from Marine Sediment, Reveals a Possible Use for Bioremediation

“…Previous studies have reported the molecular mechanism of 2-haloacid dehalogenase from Pseudomonas spp. including P. putida (Kawasaki et al 1994; Liu et al 1995; Wang et al 2018) and described known haloacid-dehalogenating bacteria and their dehalogenases (Adamu et al 2016). Of the seven genes annotated as “haloacid dehalogenase” in the SCT genome, three (locus_tag: PSCT_04345, PSCT_04409, and PSCT_04536) were SCT strain-specific.…”

Microbial Enzymes Used in Bioremediation

“…Microbial dehalogenase (EC 3.8.1.5) has attracted a great deal of attention because of its significant application in halogenated organic compounds' bioremediation. A wide range of halogenated compounds is degraded using dehalogenase enzyme, which cleaves C-X bonds [89,90] through three mechanisms, including hydrolytic, reductive, and oxygenolytic ones, which could perform dehalogenation by the replacement of the halogen atom by hydroxyl group from water and a hydrogen atom from H 2 , respectively [48].…”