Affiliation: | 1. Station Biologique de Roscoff (SBR), Sorbonne Université, CNRS, Integrative Biology of Marine Models (LBI2M), Roscoff, France Contribution: Conceptualization (equal), Formal analysis (lead), Methodology (lead), Validation (equal), Writing - original draft (lead);2. Station Biologique de Roscoff (SBR), CNRS FR2424, Sorbonne Université, Roscoff, France Contribution: Data curation (supporting), Writing - original draft (supporting), Writing - review & editing (supporting);3. Station Biologique de Roscoff (SBR), Sorbonne Université, CNRS, Integrative Biology of Marine Models (LBI2M), Roscoff, France;4. Station Biologique de Roscoff (SBR), Sorbonne Université, CNRS, Integrative Biology of Marine Models (LBI2M), Roscoff, France Contribution: Funding acquisition (lead), Supervision (equal), Validation (supporting), Writing - review & editing (supporting) |
Abstract: | Haloacid dehalogenases are potentially involved in bioremediation of contaminated environments and few have been biochemically characterized from marine organisms. The l -2-haloacid dehalogenase (l -2-HAD) from the marine Bacteroidetes Zobellia galactanivorans DsijT (ZgHAD) has been shown to catalyze the dehalogenation of C2 and C3 short-chain l -2-haloalkanoic acids. To better understand its catalytic properties, its enzymatic stability, active site, and 3D structure were analyzed. ZgHAD demonstrates high stability to solvents and a conserved catalytic activity when heated up to 60°C, its melting temperature being at 65°C. The X-ray structure of the recombinant enzyme was solved by molecular replacement. The enzyme folds as a homodimer and its active site is very similar to DehRhb, the other known l -2-HAD from a marine Rhodobacteraceae. Marked differences are present in the putative substrate entrance sites of the two enzymes. The H179 amino acid potentially involved in the activation of a catalytic water molecule was confirmed as catalytic amino acid through the production of two inactive site-directed mutants. The crystal packing of 13 dimers in the asymmetric unit of an active-site mutant, ZgHAD-H179N, reveals domain movements of the monomeric subunits relative to each other. The involvement of a catalytic His/Glu dyad and substrate binding amino acids was further confirmed by computational docking. All together our results give new insights into the catalytic mechanism of the group of marine l -2-HAD. |