L-2-Haloacid dehalogenase from Ancylobacter aquaticus UV5: Sequence determination and structure prediction

A novel 25 kDa L-2-haloacid dehalogenase (L-2-DhlB) from a recently isolated Ancylobacter aquaticus strain UV5 indigenous to contaminated site in South Africa is reported here with its gene sequence. The enzyme was purified to 22.1-fold increase in specific activity of 72.9 U/mg protein when the organism was grown in medium supplemented with 5 mM 1,2-dichloroethane (1,2-DCA). L-2-DhlB was optimally active at pH 9.0 and 37°C with poor stability at 50°C, retaining 50% of its activity after 30 min, but inactivated rapidly at 60°C. L-2-DhlB catalyzed monochloroacetate (MCA) with Km and Vmax values of 0.47 mM and 2.4 μM/min, respectively. L-2-DhlB exhibited the kcat value of 4.8/min. Expression of about 100% relative activity of L-2-DhlB on the substrate L-2-monochloropropionate (L-2-MCPA) as compared to 5% on D-2-monochloropropionate (D-2-MCPA) suggested that L-2-DhlB belongs to the family of L-2-haloacid dehalogenases. ES-mass spectroscopy and bioinformatics tools resulted in 693 bp ORF sequence corresponding to 230 amino acid protein. NCBI-BLAST of L-2-DhlB resulted in the detection of a putative conserved domain of hypothetical haloacid dehalogenase (HAD)-like superfamily and subfamily IA.