Characterization of the haloacid dehalogenase from Xanthobacter autotrophicus GJ10 and sequencing of the dhlB gene

Abstract

The haloacid dehalogenase of the 1,2-dichloroethane-utilizing bacterium Xanthobacter autotrophicus GJ1O was purified from a mutant with an eightfold increase in expression of the enzyme. The mutant was obtained by selecting for enhanced resistance to monobromoacetate. The enzyme was purified through (NH4)2S04 fractionation, DEAE-cellulose chromatography, and hydroxylapatite chromatography. The Hydrolytic dehalogenases are key enzymes in the detoxification of aliphatic halogenated hydrocarbons. They catalyze the cleavage of carbon-halogen bonds through a nucleophilic substitution by water to yield an alcohol. At least two distinct groups can be recognized with respect to their substrate ranges: haloalkane dehalogenases hydrolyze halogenated alkanes, whereas haloacid dehalogenases are active with short-chain 2-halogenated carboxylic acids.Of the 2-haloacid dehalogenases (E.C. 3.8.1.2), a number of enzymes have been purified and characterized (14,16,17,19,22,29,34). They have been divided in different classes according to their substrate specificity (9), electrophoretic mobility on polyacrylamide gels (9, 36), and stereospecific action on 2-monochloropropionic acid (2-MCPA) (36). Four different types of dehalogenation of 2-MCPA can be recognized. Two of these are represented by enzymes that are active with only L-or D-2-MCPA, giving products with inverted configuration at the chiral carbon atom. The other two act on both isomers, one with inversion of configuration and the other with retention of configuration.Previous studies have shown the presence of more than one haloacid dehalogenase in the same organism (9, 16), with only minor differences in substrate specificities. It has been suggested that these isoenzymes have arisen by gene duplication and subsequent divergent evolution (9).We are interested in the relation between structure, enzymatic mechanism, and evolution of dehalogenases. To study these aspects, we started to investigate the haloacid dehalogenase of Xanthobacter autotrophicus GJ1O. This bacterium was isolated on 1,2-dichloroethane as the sole carbon and energy source (13 In a previous paper, we reported the cloning of both dehalogenase genes from GJ10 (12). The haloalkane dehalogenase has been purified (15) and crystallized (25), and its sequence (12) and tertiary structure (5) have been determined. The haloacid dehalogenase gene has been cloned on a 10-kb fragment in the broad-host-range cosmid vector pLAFR1 (12). Here, we describe the purification and characterization of the haloacid dehalogenase from an overproducing mutant of GJ10 and the sequence of the gene encoding this enzyme. The properties of the enzyme are compared with those of other haloacid dehalogenases and haloalkane dehalogenase. MATERIALS AND METHODSGrowth conditions. Strains and plasmids used are listed in

Crystallization and preliminary X‐ray analysis of L‐2‐haloacid dehalogenase from xanthobacter autotrophicus GJ10

“…The amino acid sequence is more than 40% identical to six other L-specific 2-haloacid dehalogenases from different sources (Kawasaki et al, 1994; Liu et al, 1994), but it shows no homology to the haloalkane dehalogenase from X . autotrophicus (Van der Ploeg et al, 1991). The enzyme shows maximum activity at pH 9.5.…”

“…autotrophicus belongs to the group of L-specific dehalogenases. The dhlB gene encoding for it was cloned and sequenced (Van der Ploeg et al, 1991).The protein consists of a single polypeptide chain of 253 amino acids and has a molecular weight of 27,558 Da. The amino acid sequence is more than 40% identical to six other L-specific 2-haloacid dehalogenases from different sources (Kawasaki et al, 1994; Liu et al, 1994), but it shows no homology to the haloalkane dehalogenase from X .…”

“…YL. To gain more insight into the factors that determine the substrate specificity and stereospecific catalysis and to obtain structural evidence for one of the possible reaction mechanisms, we extended our crystallographic studies on dehalogenating enzymes to the L-2-haloacid dehalogenase.L-2-Haloacid dehalogenase was prepared as described (Van der Ploeg et al, 1991). Crystals were grown at room temperature by vapor diffusion in hanging drops (3 pL of protein and 3 pL of precipitant) suspended over a 1-mL reservoir of precipitant.…”

Crystal Structures of Intermediates in the Dehalogenation of Haloalkanoates by l-2-Haloacid Dehalogenase

“…DhlB is somewhat larger, and the 21 extra residues form a two-helix excursion from the ␣/␤ core domain on the same side as the four-helix bundle. Together these helical domains provide a tight dimer interface and limit the substrate specificity of the X. autotrophicus enzyme to short substrates such as haloacetates and halopropionates (8,9).Comprehensive biochemical data have been obtained for the Pseudomonas enzyme (1, 10, 11).2 Asp 8 was identified as the nucleophile in the first step of the enzymatic reaction, the formation of a covalent enzyme-ester intermediate. ) that are involved in substrate binding and catalysis.…”

Biochemical and structural characterisation of a haloalkane dehalogenase from a marine Rhodobacteraceae

“…The HLDs from the bacteria Xanthobacter autotrophicus (DhlA) [3,4], Rhodococcus rhodochrous (DhaA) [5], Sphingomonas paucimobilis (LinB) [6,7], Bradyrhizobium japonicum USDA110 (DbjA) [8,9], Mycobacterium tuberculosis (DmbC) [10] and Plesiocystis pacifica SIR-1 (DppA) [11] have been biochemically and structurally characterised and are two domain proteins. The core domain belongs to the a/b hydrolase protein fold family with the active site located on the interface of the core and the cap domain, the latter determining substrate specificity.…”