(1) Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York, YO10 5YW, UK;(2) Henry Wellcome Building for Biocatalysis, School of Biosciences, University of Exeter, Stocker Road, Exeter, EX4 4QD, UK
Abstract:
Haloacid dehalogenases have potential applications in the pharmaceutical and fine chemical industry as well as in the remediation
of contaminated land. The l-2-haloacid dehalogenase from the thermophilic archaeon Sulfolobus tokodaii has been cloned and over-expressed in Escherichia coli and successfully purified to homogeneity. Here we report the structure of the recombinant dehalogenase solved by molecular
replacement in two different crystal forms. The enzyme is a homodimer with each monomer being composed of a core-domain of
a β-sheet bundle surrounded by α-helices and an α-helical sub-domain. This fold is similar to previously solved mesophilic
l-haloacid dehalogenase structures. The monoclinic crystal form contains a putative inhibitor l-lactate in the active site. The enzyme displays haloacid dehalogenase activity towards carboxylic acids with the halide attached
at the C2 position with the highest activity towards chloropropionic acid. The enzyme is thermostable with maximum activity
at 60°C and a half-life of over 1 h at 70°C. The enzyme is relatively stable to solvents with 25% activity lost when incubated
for 1 h in 20% v/v DMSO.