Cloning, Biochemical Properties, and Distribution of Mycobacterial Haloalkane Dehalogenases

Abstract

Haloalkane dehalogenases are enzymes that catalyze the cleavage of the carbon-halogen bond by a hydrolytic mechanism. Genomes of Mycobacterium tuberculosis and M. bovis contain at least two open reading frames coding for the polypeptides showing a high sequence similarity with biochemically characterized haloalkane dehalogenases. We describe here the cloning of the haloalkane dehalogenase genes dmbA and dmbB from M. bovis 5033/66 and demonstrate the dehalogenase activity of their translation products. Both of these genes are widely distributed among species of the M. tuberculosis complex, including M. bovis, M. bovis BCG, M. africanum, M. caprae, M. microti, and M. pinnipedii, as shown by the PCR screening of 48 isolates from various hosts. DmbA and DmbB proteins were heterologously expressed in Escherichia coli and purified to homogeneity. The DmbB protein had to be expressed in a fusion with thioredoxin to obtain a soluble protein sample. The temperature optimum of DmbA and DmbB proteins determined with 1,2-dibromoethane is 45°C. The melting temperature assessed by circular dichroism spectroscopy of DmbA is 47°C and DmbB is 57°C. The pH optimum of DmbA depends on composition of a buffer with maximal activity at 9.0. DmbB had a single pH optimum at pH 6.5. Mycobacteria are currently the only genus known to carry more than one haloalkane dehalogenase gene, although putative haloalkane dehalogenases can be inferred in more then 20 different bacterial species by comparative genomics. The evolution and distribution of haloalkane dehalogenases among mycobacteria is discussed.Haloalkane dehalogenases catalyze detoxification reactions and act on a broad range of halogenated aliphatic compounds (15). Haloalkane dehalogenases have primarily been isolated from bacterial strains living in soil contaminated with halogenated aliphatic compounds (16,20,30,37,38,41,42,45). The enzymes are involved in biochemical pathways enabling bacteria to utilize halogenated compounds, i.e., 1,2-dichloroethane, 1-chloroalkanes, ␥-hexachlorocyclohexane, 1,2-dibromoethane, and 1,3-dichloropropene as the source of carbon and energy.Database searches revealed the presence of at least two open reading frames, rv2579 and rv2296, with sequences highly similar to haloalkane dehalogenases in the genome of the pathogenic bacterium Mycobacterium tuberculosis H37Rv (4). Dehalogenating activity was consequently confirmed in M. tuberculosis H37Rv (19). The same study demonstrated dehalogenase activity in several saprophytic mycobacterial species. The first haloalkane dehalogenase originating from a mycobacterial strain was cloned from Mycobacterium sp. strain GP1 (37). This haloalkane dehalogenase, designated DhaA f , is involved in the biochemical pathway for biodegradation of 1,2-dibromoethane. Genome sequence analysis has shown that two other representatives (fast-growing M. smegmatis MC2 155 and slowgrowing M. avium subsp. paratuberculosis K10), possess open reading frames aal17946, map0345c, and map2057 coding for putative haloalkane dehalogenases....

Isolation and characterization of monochloroacetic acid-degrading bacteria

“…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).…”

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

“…The dehalogenation of chlorinated alcohol ethers by these enzymes, however, may be a novel reaction. The involvement of multiple dehalogenases in the same strain also cannot be ruled out completely (7,9).…”

“…In some cases, however, enzymatic reactions may facilitate transformation of the hemiacetal (17). An O-dealkylation mechanism has been suggested for BCEE degradation by a Rhodococcus strain (9) and Rhodococcus strain DTB (12). In the case of chlorinated ethers like BCEE, however, additional biological reactions may be required prior to ether scission.…”

Discovery of Novel Haloalkane Dehalogenase Inhibitors

“…obvious (15,39,40), (iii) genetic engineering of mycobacteria is complicated due to the extremely slow growth and high pathogenicity of the organisms, and (iv) it was the enzyme least affected by the ligand-based inhibitors.…”

“…For instance, it is undoubtedly interesting that the plant pathogen Agrobacterium tumefaciens C58 carries the HLDencoding gene datA on its tumor-inducing plasmid while there is no clear link between HLD activity and tumorigenesis (14). Similarly, the presence of three different HLD genes in the genome of the human pathogen Mycobacterium tuberculosis H37Rv (15) suggests that HLDs are important for its survival, but it is not immediately obvious why an organism that colonizes human tissues would require enzymes that cleave carbon-halogen bonds. One way to study the natural function of an enzyme is to use specific inhibitors.…”