RESUMO
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 beta-sheet bundle surrounded by alpha-helices and an alpha-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 degrees C and a half-life of over 1 h at 70 degrees C. The enzyme is relatively stable to solvents with 25% activity lost when incubated for 1 h in 20% v/v DMSO.
Assuntos
Hidrolases/metabolismo , Sulfolobus/enzimologia , Sequência de Aminoácidos , Cristalografia por Raios X , Estabilidade Enzimática , Escherichia coli/genética , Temperatura Alta , Hidrolases/química , Hidrolases/genética , Hidrolases/isolamento & purificação , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The L-2-haloacid dehalogenase enzymes catalyse the hydrolytic cleavage of a halogen from the C2 position of short-chain haloacids. The recombinant dehalogenase from the thermophilic archaeon Sulfolobus tokodaii has been cloned, overexpressed and purified to homogeneity. The 24 kDa enzyme was crystallized using the microbatch method in the monoclinic space group C2, with unit-cell parameters a = 127.6, b = 58.1, c = 51.2 A, beta = 97.2 degrees . Data were collected to 1.9 A resolution using synchrotron radiation and the structure was solved by molecular replacement. Analysis of the data and the preliminary refined model showed that the crystal was an order-disorder twin by reticular merohedry with a twin index of 10. It was possible to de-twin the experimental data utilizing the symmetry of the molecular layers from which the crystal is built.