The Crystal Structure of DehI Reveals a New α-Haloacid Dehalogenase Fold and Active-Site Mechanism

Abstract

Haloacid dehalogenases catalyse the removal of halides from organic haloacids and are of interest for bioremediation and for their potential use in the synthesis of industrial chemicals. We present the crystal structure of the homodimer DehI from Pseudomonas putida strain PP3, the first structure of a group I α-haloacid dehalogenase that can process both l- and d-substrates. The structure shows that the DehI monomer consists of two domains of ∼ 130 amino acids that have ∼ 16% sequence identity yet adopt virtually identical and unique folds that form a pseudo-dimer. Analysis of the active site reveals the likely binding mode of both l- and d-substrates with respect to key catalytic residues. Asp189 is predicted to activate a water molecule for nucleophilic attack of the substrate chiral centre resulting in an inversion of configuration of either l- or d-substrates in contrast to d-only enzymes. These details will assist with future bioengineering of dehalogenases.

Introduction

Industrial-scale manufacturing and chemical processing is responsible for the introduction of vast amounts of xenobiotic compounds into the biosphere. While the metabolic versatility of microbial communities is enormous, many xenobiotics possess chemical modifications that can prevent their efficient degradation. For instance, halogen substitution often increases the recalcitrance and toxicity of an organic molecule,¹ and thus, halogenated organic compounds, which represent a significant subset of industrially important solvents, herbicides, and pesticides, have been the targets of bioremedial research projects aimed at their removal from contaminated systems.

Dehalogenases are microbial enzymes that catalyse the critical step in the breakdown of priority halogenated organic pollutants—cleavage of the carbon–halogen bond.² Among these, α-haloacid (αHA) dehalogenases have been extensively studied and are divided into two phylogenetic groups, I and II.³ Both groups are active on low molecular weight organic acids halogenated at the C^α position,⁴ and both utilise a hydrolytic S_N2 substitution reaction, which results in inversion of the substrate configuration. Where the groups differ is in enantiomer selectivity and in the nature of the hydrolytic attack. Group II αHA dehalogenases, which are the better characterised both biochemically and structurally,5, 6, 7, 8, 9, 10, 11 have been shown to specifically degrade l-haloacid substrate molecules. The reaction involves two nucleophilic attacks, first at the C^α atom of the substrate molecule by an aspartate residue, forming an esterified intermediate (displacing the halide), then by an activated water molecule, which attacks the aspartate C^γ atom, to cleave the enzyme-product ester bond.12, 13

The group I αHA dehalogenases have no reported sequence similarity with other proteins and, up to this report, no structural information has been available. Some group I αHA dehalogenases are able to process both l- and d-haloacids,14, 15 whilst others can only process the d-enantiomer. Studies of the group I αHA dehalogenase, DL-DEX from Pseudomonas sp. strain 113, have led to the identification of functionally important residues¹⁶ and have shown that hydrolysis of the substrate proceeds through a direct attack of a water molecule on the α-carbon of l- or d-2-haloalkanoic acids, displacing the halogen atom.¹⁷ Thus, the group I αHA dehalogenases represent the first case of a hydrolytic dehalogenation reaction that does not involve a covalent ester intermediate.

We report the crystal structure of DehI, a representative member of the group I αHA dehalogenases, from Pseudomonas putida strain PP3, of interest for its potential in biodegradation. Strain PP3 was originally isolated from chemostat culture following selection on the herbicide Dalapon (2,2-dichloropropionic acid),¹⁸ which is considered a priority pollutant in many countries around the world. The enzyme consists of 296 amino acids with a molecular mass of 32.7 kDa. The structure provides details of a new fold and of the substrate-binding site allowing the role of key functional residues to be resolved. The structure also provides insight into the reaction mechanism of DehI and its ability to process both l- and d-substrates, in contrast to closely related homologues that only turn over d-substrates.