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Recently, we found that Alcanivorax bacteria from various marine environments were capable of degrading halogenated alkanes. Genome sequencing of A. dieselolei B-5 revealed two putative haloalkane dehalogenase (HLD) genes, which were supposed to be involved in degradation of halogenated compounds. In this report, we confirm for the first time that the Alcanivorax bacterium encodes a truly functional HLD named DadB. An activity assay with 46 halogenated substrates indicated that DadB possesses broad substrate range and has the highest overall activity among the identified HLDs. DadB prefers brominated substrates; chlorinated alkenes; and the C2-C3 substrates, including the persistent pollutants of 1,2-dichloroethane, 1,2-dichloropropane and 1,2,3-trichloropropane. As DadB displays no detectable activity toward long-chain haloalkanes such as 1-chlorohexadecane and 1-chlorooctadecane, the degradation of them in A. dieselolei B-5 might be attributed to other enzymes. Kinetic constants were determined with 6 substrates. DadB has highest affinity and largest kcat/Km value toward 1,3-dibromopropane (Km = 0.82 mM, kcat/Km = 16.43 mM−1·s−1). DadB aggregates fast in the buffers with pH≤7.0, while keeps stable in monomer form when pH≥7.5. According to homology modeling, DadB has an open active cavity with a large access tunnel, which is supposed important for larger molecules as opposed to C2-C3 substrates. Combined with the results for other HLDs, we deduce that residue I247 plays an important role in substrate selection. These results suggest that DadB and its host, A. dieselolei B-5, are of potential use for biocatalysis and bioremediation applications. [ABSTRACT FROM AUTHOR]

Haloalkane dehalogenases are key enzymes for biodegradation of halogenated aliphatic compounds, widely distributed in various microbial species of wide geographical distributions, and of significance in practical application such as bioremediation and industrial biocatalysis. Twenty haloalkane dehalogenases have been biochemically characterized so far. In recent years, progresses have been made in the enzymatic characteristics, protein structures, and phylogenetic diversity. We reviewed the progresses of haloalkane dehalogenase in structure and function, diversity and application potential.

[OBJECTIVE] Alcanivorax dieselolei B-5 is an important oil-degrading bacterium. We studied its substrate range and degradation of halogenated compounds. [METHODS] Growth capability of B-5 was examined with different halogenated substrates as sole carbon source. A putative haloalkane dehalogenase (HLD) gene named dadA was found from the genome of strain B-5 and analyzed by sequence alignment, phylogenetic analysis and homologous modeling. After heterologous expression in Escherichia coli and purification, the activity of DadA towards 46 substrates was determined. [RESULTS] Strain B-5 was capable of utilizing various halogenated compounds (C3-C,8) as the sole carbon source. DadA had typical catalytic pentad residues of HLD-II subfamily, but it was independent from other members of this subfamily according to phylogenetic analysis. Activity assay showed that DadA has higher specificity and narrower substrate range than other characterized HLDs and it only showed activity toward 1,2,3-tribromopropane, 1,2-dibromo-3-chloropropane and 2,3-dichloroprop-1-ene among 46 tested substrates. [CONCLUSIONS] Strain B-5 and its HLD DadA can degrade halogenated aliphatic pollutants although.