Two Rhizobial Strains,Mesorhizobium lotiMAFF303099 andBradyrhizobium japonicumUSDA110, Encode Haloalkane Dehalogenases with Novel Structures and Substrate Specificities

Abstract

Haloalkane dehalogenases are key enzymes for the degradation of halogenated aliphatic pollutants. Two rhizobial strains, Mesorhizobium loti MAFF303099 and Bradyrhizobium japonicum USDA110, have open reading frames (ORFs), mlr5434 and blr1087, respectively, that encode putative haloalkane dehalogenase homologues. The crude extracts of Escherichia coli strains expressing mlr5434 and blr1087 showed the ability to dehalogenate 18 halogenated compounds, indicating that these ORFs indeed encode haloalkane dehalogenases. Therefore, these ORFs were referred to as dmlA (dehalogenase from Mesorhizobium loti) and dbjA (dehalogenase from Bradyrhizobium japonicum), respectively. The principal component analysis of the substrate specificities of various haloalkane dehalogenases clearly showed that DbjA and DmlA constitute a novel substrate specificity class with extraordinarily high activity towards ␤-methylated compounds. Comparison of the circular dichroism spectra of DbjA and other dehalogenases strongly suggested that DbjA contains more ␣-helices than the other dehalogenases. The dehalogenase activity of resting cells and Northern blot analyses both revealed that the dmlA and dbjA genes were expressed under normal culture conditions in MAFF303099 and USDA110 strain cells, respectively.Haloalkane dehalogenases are key enzymes for the degradation of halogenated aliphatic compounds that occur as soil pollutants (1,17,38). Haloalkane dehalogenases catalyze the hydrolytic cleavage of the carbon-halogen bond(s) and produce the corresponding alcohols, halide ions, and protons. These enzymes belong to the ␣/␤-hydrolase superfamily (39).Haloalkane dehalogenases are attractive targets for proteinengineering studies aimed at improving catalytic efficiency and at broadening the range of substrate specificity for important environmental pollutants. To date, the three-dimensional structures of three haloalkane dehalogenases have been determined by protein crystallography: DhlA from Xanthobacter autotrophicus GJ10 (48), DhaA from Rhodococcus sp. (25, 35), and LinB from Sphingomonas paucimobilis UT26 (29). The differences in the substrate specificities of these three haloalkane dehalogenases can be accounted for on the basis of their three-dimensional structures (11). Comparison of the kinetic mechanisms of DhlA, DhaA, and LinB showed that the overall reaction mechanisms are similar but that the rate-limiting steps differ, i.e., halide release in the case of DhlA (44), liberation of an alcohol in the case of DhaA (4), and hydrolysis of an alkyl-enzyme intermediate in the case of LinB (41). Partial improvement in the catalytic properties and modification of the substrate specificities of haloalkane dehalogenases by rational design (5, 34) and directed evolution approaches (3, 40) have recently been reported. However, it remains difficult to construct mutant enzymes with entirely new capabilities using only protein-engineering techniques, and therefore, the isolation of new family members is still desirable.For quite some time, haloalkane ...

Isolation and characterization of monochloroacetic acid-degrading bacteria

“…The enzyme activities of the haloalkane dehalogenases toward various substrates have been assessed, and it was reported that DbjA showed higher dehalogenation activity for substrates carrying a bulky substituent at the β position than did LinB (Sato et al, 2005). Such information on substrate specifi city will provide clues on ways to alter the substrate specifi city of this unique class of enzymes.…”

“…Although there is only limited information on the diversity of bacterial haloacetic acid dehalogenases, the homologous proteins (or the corresponding genes) of haloalkane dehalogenases LinB from Sphingobium japonicum strain UT26 (Nagata et al, 1993) have been found in various bacteria, including Mesorhizobium loti strain MAFF303099 (DmlA), Bradyrhizobium japonicum USDA110 (DbjA) (Sato et al, 2005), and Mycobacterium avium strain N85 (DhmA) (Jesenska et al, 2005). The enzyme activities of the haloalkane dehalogenases toward various substrates have been assessed, and it was reported that DbjA showed higher dehalogenation activity for substrates carrying a bulky substituent at the β position than did LinB (Sato et al, 2005).…”

Joint Improvised Explosive Device Defeat Organization: Anomaly or Future Roadmap

“…2A). Several haloalkane dehalogenases studied to date have been shown to have broad substrate ranges (6,15), and to dehalogenate chlorinated ethers (6,26) and alcohols (9). The dehalogenation of a chlorinated alcohol ether by these enzymes, however, may be a novel reaction.…”

Biodegradation of Bis(2-Chloroethyl) Ether by Xanthobacter sp. Strain ENV481

“…2A). Several haloalkane dehalogenases studied to date have been shown to have broad substrate ranges (7,16) and to dehalogenate chlorinated ethers (7,27) and alcohols (11). The dehalogenation of chlorinated alcohol ethers by these enzymes, however, may be a novel reaction.…”