title: Biochemical and biophysical characterisation of haloalkane dehalogenases DmrA and DmrB in Mycobacterium strain JS60 and their role in growth on haloalkanes.
abstract: Haloalkane dehalogenases (HLDs) catalyse the hydrolysis of haloalkanes to alcohols, offering a biological solution for toxic haloalkane industrial wastes. Hundreds of putative HLD genes have been identified in bacterial genomes, but relatively few enzymes have been characterised. We identified two novel HLDs in the genome of Mycobacterium rhodesiae strain JS60, an isolate from an organochlorine-contaminated site: DmrA and DmrB. Both recombinant enzymes were active against C2-C6 haloalkanes, with a preference for brominated linear substrates. However, DmrA had higher activity against a wider range of substrates. The kinetic parameters of DmrA with 4-bromobutyronitrile as a substrate were Km  = 1.9 ± 0.2 mM, kcat  = 3.1 ± 0.2 s(-1) . DmrB showed the highest activity against 1-bromohexane. DmrA is monomeric, whereas DmrB is tetrameric. We determined the crystal structure of selenomethionyl DmrA to 1.7 Å resolution. A spacious active site and alternate conformations of a methionine side-chain in the slot access tunnel may contribute to the broad substrate activity of DmrA. We show that M. rhodesiae JS60 can utilise 1-iodopropane, 1-iodobutane and 1-bromobutane as sole carbon and energy sources. This ability appears to be conferred predominantly through DmrA, which shows significantly higher levels of upregulation in response to haloalkanes than DmrB.
authors.affiliation: School of Molecular Bioscience, University of Sydney, Sydney, NSW, 2006, Australia.
authors.forename: Herman K H
authors.initials: HK
authors.lastname: Fung
authors.affiliation: School of Molecular Bioscience, University of Sydney, Sydney, NSW, 2006, Australia.
authors.forename: Morgan S
authors.initials: MS
authors.lastname: Gadd
authors.affiliation: School of Molecular Bioscience, University of Sydney, Sydney, NSW, 2006, Australia.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Drury
authors.affiliation: School of Molecular Bioscience, University of Sydney, Sydney, NSW, 2006, Australia.
authors.forename: Samantha
authors.initials: S
authors.lastname: Cheung
authors.affiliation: School of Molecular Bioscience, University of Sydney, Sydney, NSW, 2006, Australia.
authors.forename: J Mitchell
authors.initials: JM
authors.lastname: Guss
authors.affiliation: School of Molecular Bioscience, University of Sydney, Sydney, NSW, 2006, Australia.
authors.forename: Nicholas V
authors.initials: NV
authors.lastname: Coleman
authors.affiliation: School of Molecular Bioscience, University of Sydney, Sydney, NSW, 2006, Australia.
authors.forename: Jacqueline M
authors.initials: JM
authors.lastname: Matthews
mesh_terms: D000473:Alkanes / Q000378:metabolism*; D002244:Carbon / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004734:Energy Metabolism; D004783:Environmental Microbiology; D006846:Hydrocarbons, Halogenated / Q000378:metabolism*; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000201:enzymology* / Q000254:growth & development / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
chemical_list: D000473:Alkanes; D004269:DNA, Bacterial; D006846:Hydrocarbons, Halogenated; D002244:Carbon; D006867:Hydrolases; C046137:haloalkane dehalogenase