Two rhizobial strains, Mesorhizobium loti MAFF303099 and Bradyrhizobium japonicum USDA110, encode haloalkane dehalogenases with novel structures and substrate specificities - PubMed (original) (raw)

Two rhizobial strains, Mesorhizobium loti MAFF303099 and Bradyrhizobium japonicum USDA110, encode haloalkane dehalogenases with novel structures and substrate specificities

Yukari Sato et al. Appl Environ Microbiol. 2005 Aug.

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 beta-methylated compounds. Comparison of the circular dichroism spectra of DbjA and other dehalogenases strongly suggested that DbjA contains more alpha-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.

PubMed Disclaimer

Figures

FIG. 1.

Alignment of amino acid sequences of the putative dehalogenases from M. loti MAFF303099 (Mlr5434/DmlA) and B. japonicum USDA110 (Blr1087/DbjA) and haloalkane dehalogenase LinB from S. paucimobilis UT26. Putative protein products of mlr5434 (Mlr5434) and blr1087 (Blr1087) were named DmlA and DbjA, respectively. Sequence alignment was created using CLUSTALW 1.7 (47) and adjusted manually. The secondary structure elements (indicated by lines under the sequence) and the catalytic triad (indicated by triangles above the sequence) of LinB were deduced from the crystal structure (29). Secondary structure elements of DbjA and DmlA are consensus predictions using the programs PHD (43), PSIPRED (20), Jpred (9), SSThread (15), and Network Protein Sequence Analysis (8).

FIG. 2.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses of Trx-DmlA (a), DbjA (b), and His-tagged DbjA (c). a. E. coli BL21(DE3)(pYMLA2)(pG-KJE8) cells. Lanes: 1, total proteins of noninduced cells; 2, total proteins of IPTG-induced cells; 3, crude extract of IPTG-induced cells. b. E. coli BL21(pYBJA1) cells. Lanes: 1, total proteins of noninduced cells; 2, total proteins of IPTG-induced cells; 3, crude extract of IPTG-induced cells. c. Protein patterns during the His-tagged DbjA purification from E. coli BL21(pYBJA2) cells. Lanes: 1, total proteins of noninduced cells; 2, total proteins of IPTG-induced cells; 3, crude extract of IPTG-induced cells; 4, purified His-tagged DbjA. Arrows indicate Trx-DmlA (a), DbjA (b), and His-tagged DbjA (c), respectively.

FIG. 3.

CD spectra of haloalkane dehalogenases. a. Far-UV CD spectra of four haloalkane dehalogenases, LinB, DhaA, DhlA, and DbjA. b. Comparison of α-helical content of haloalkane dehalogenases estimated by the self-consistent method (45). The predictions made using this method corresponded well with the secondary structure content deduced from the crystal structure (LinB, 40.3% of α-helical content; DhaA, 43% of α-helical content; DhlA, 40.9% of α-helical content) (35, 37, 42).

FIG. 4.

Principal component analysis of substrate specificities of DbjA, DmlA, and other haloalkane dehalogenases. (a and b) The score plot (a) and the loading plot (b) of the first principal component from analysis of specific activities determined for 16 halogenated substrates.

References

1. Ballschmiter, K. 2003. Pattern and sources of naturally produced organohalogens in the marine environment: biogenic formation of organohalogens. Chemosphere 52:313-324. - PubMed
1. Beringer, J. E. 1974. R factor transfer in Rhizobium leguminosarum. J. Gen. Microbiol. 84:188-198. - PubMed
1. Bosma, T., J. Damborsky, G. Stucki, and D. B. Janssen. 2002. Biodegradation of 1,2,3-trichloropropane through directed evolution and heterologous expression of a haloalkane dehalogenase gene. Appl. Environ. Microbiol. 68:3582-3587. - PMC - PubMed
1. Bosma, T., M. G. Pikkemaat, J. Kingma, J. Dijk, and D. B. Janssen. 2003. Steady-state and pre-steady-state kinetic analysis of halopropane conversion by a rhodococcus haloalkane dehalogenase. Biochemistry 42:8047-8053. - PubMed
1. Chaloupkova, R., J. Sykorova, Z. Prokop, A. Jesenska, M. Monincova, M. Pavlova, M. Tsuda, Y. Nagata, and J. Damborsky. 2003. Modification of activity and specificity of haloalkane dehalogenase from Sphingomonas paucimobilis UT26 by engineering of its entrance tunnel. J. Biol. Chem. 52:52622-52628. - PubMed

Two rhizobial strains, Mesorhizobium loti MAFF303099 and Bradyrhizobium japonicum USDA110, encode haloalkane dehalogenases with novel structures and substrate specificities - PubMed (original) (raw)