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Biochemical and biophysical characterisation of haloalkane dehalogenases DmrA and DmrB in M ycobacterium strain JS 60 and their role in growth on haloalkanes
Author(s) -
Fung Herman K. H.,
Gadd Morgan S.,
Drury Thomas A.,
Cheung Samantha,
Guss J. Mitchell,
Coleman Nicholas V.,
Matthews Jacqueline M.
Publication year - 2015
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/mmi.13039
Subject(s) - enzyme , biology , stereochemistry , hydrolysis , substrate (aquarium) , active site , strain (injury) , monomer , biochemistry , hydrolase , chemistry , organic chemistry , ecology , polymer , anatomy
Summary 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 M ycobacterium rhodesiae strain JS60, an isolate from an organochlorine‐contaminated site: DmrA and DmrB . Both recombinant enzymes were active against C 2– C 6 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 K m = 1.9 ± 0.2 mM, k cat = 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 JS 60 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 .