Journal of Molecular Biology
The Crystal Structure of DehI Reveals a New α-Haloacid Dehalogenase Fold and Active-Site Mechanism
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 Cα position,4 and both utilise a hydrolytic SN2 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 Cα 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 Cγ 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.
Section snippets
Crystal structure of DehI
The crystallographic structure of the DehI homodimer is the first reported structure of a group I αHA dehalogenase (Fig. 1a). The monomer (Fig. 1b) is highly α-helical and is composed of a repeated motif, which is likely to have arisen from gene duplication. The repeats (residues 1-130 and 166-296) share only 16% sequence identity yet can be superposed with an RMSD of 1.67 Å (116 out of 130 Cα atoms) (Fig. 1c and d). Each repeat is composed of six α-helices (repeat 1, α-helices 1–6; repeat 2,
Protein expression, purification, and crystallization
The gene encoding DehI was cloned into a pET15b vector (Novagen) featuring an N-terminal Hexa-His tag. The protein was overexpressed in Escherichia coli Nova Blue (DE3) and purified using three chromatographic steps, IMAC beads (BIORAD), charged with nickel, Q-Sepharose (Pharmacia/Pfizer, New York, NY), and Superdex 75 (Pharmacia/Pfizer). The purified DehI was concentrated to ∼ 8 mg/ml estimated using A280 readings (6-ml Vivaspin concentrator tube with 10,000-Da molecular mass cutoff;
Protein Data Bank accession code
The coordinates and structure factors have been deposited in the Protein Data Bank under code 3BJX.
Acknowledgements
J.W.S. is supported by a University of Western Australia postgraduate scholarship. M.C.J.W. is a National Health and Medical Research Council Senior Research Fellow and acknowledges the support of the Australian Research Council and the National Health and Medical Research Council. J.A.W. acknowledges the support of the Australian Research Council. J.C.W. is a National Health and Medical Research Council Principal Research Fellow. A.J.W. gratefully acknowledges the support of the Royal Society.
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