Biochemical Characterization of a Haloalkane Dehalogenase DadB from Alcanivorax dieselolei B-5

Abstract

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 k cat/K m value toward 1,3-dibromopropane (K m = 0.82 mM, k cat/K m = 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.

Ancestral Haloalkane Dehalogenases Show Robustness and Unique Substrate Specificity

“…[34][35][36] During the past three decades, 23 HLDs have been identified and (at least partially) biochemically characterized. [37][38][39][40][41][42] The majority of mechanistic and structural information has been obtained for enzymesf rom the HLD-II subfamily.…”

Hydrocarbonoclastic Alcanivorax Isolates Exhibit Different Physiological and Expression Responses to n-dodecane

“…dieselolei can degrade a wide spectrum of linear and branched alkanes (Liu and Shao, 2005; Wang and Shao, 2014), while A. borkumensis can degrade linear alkanes, cycloalkanes and isoprenoids (Yakimov et al, 1998). Many genes encoding for enzymes initiating the degradation of hydrocarbons have been detected in the genomes of the two species: alk B and p450 are present in both, while alm A and dad B are peculiar of A. dieselolei (Sabirova et al, 2006, 2011; Wang et al, 2010; Liu et al, 2011; Wang and Shao, 2012, 2014; Li and Shao, 2014). As many other hydrocarbonoclastic bacteria, they are able to produce biosurfactants to facilitate their access to alkanes.…”

RNA‐seq transcriptome analysis of a Pseudomonas strain with diversified catalytic properties growth under different culture medium

“…; Frusciante et al. ; Li and Shao ). Nowadays, RNA‐seq, the next‐generation technique in sequencing of RNA, gives unprecedented detail about the transcriptional landscape of an organism (Young et al.…”

“…However, there are still many restrictions for widespread application of large-scale biocatalysis in chiral pharmaceuticals, especially for the limited commercially available bioenzymes and the low yields of products . In recent years, the application of genomic sequencing was been found to be greatly helpful for screening target genes encoding expected enzymes from a whole-cell microorganism biocatalyst (Chen et al 2014a;Frusciante et al 2014;Li and Shao 2014). Nowadays, RNA-seq, the next-generation technique in sequencing of RNA, gives unprecedented detail about the transcriptional landscape of an organism (Young et al 2010).…”