Fung Herman K H, Gadd Morgan S, Drury Thomas A, Cheung Samantha, Guss J Mitchell, Coleman Nicholas V, Matthews Jacqueline M
School of Molecular Bioscience, University of Sydney, Sydney, NSW, 2006, Australia.
Mol Microbiol. 2015 Aug;97(3):439-53. doi: 10.1111/mmi.13039. Epub 2015 May 20.
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.
卤代烷脱卤酶（HLDs）催化卤代烷水解生成醇类，为有毒卤代烷工业废物提供了一种生物处理方法。在细菌基因组中已鉴定出数百个推定的HLD基因，但得到表征的酶相对较少。我们在罗得西亚分枝杆菌菌株JS60的基因组中鉴定出两种新型HLDs，该菌株是从一个有机氯污染场地分离得到的：DmrA和DmrB。两种重组酶对C2 - C6卤代烷均有活性，更倾向于溴代线性底物。然而，DmrA对更广泛的底物具有更高的活性。以4 - 溴丁腈为底物时，DmrA的动力学参数为Km = 1.9 ± 0.2 mM，kcat = 3.1 ± 0.2 s(-1)。DmrB对1 - 溴己烷表现出最高活性。DmrA是单体，而DmrB是四聚体。我们将硒代甲硫氨酸标记的DmrA晶体结构解析到了1.7 Å的分辨率。一个宽敞的活性位点以及在狭槽通道中蛋氨酸侧链的交替构象可能有助于DmrA具有广泛的底物活性。我们发现罗得西亚分枝杆菌JS60能够利用1 - 碘丙烷、1 - 碘丁烷和1 - 溴丁烷作为唯一的碳源和能源。这种能力似乎主要是由DmrA赋予的，与DmrB相比，DmrA对卤代烷的响应上调水平显著更高。
