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

• 25 kDa l-2-haloacid dehalogenase from Ancylobacter aquaticus strain UV5 is reported.
• Enzyme was purified to 22.1-fold increase in specific activity of 72.9 U/mg protein.
• The enzyme was optimally active at pH 9.0 and temperature 37 °C.
• Enzyme showed Km = 0.47 mM, Vmax = 2.4 μM/min and kcat = 4.8/min for monochloroacetate.
• l-2-Haloacid dehalogenase is of 693 bp DNA corresponding to 230 amino acid protein.

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.