Construction and characterization of histidine-tagged haloalkane dehalogenase (LinB) of a new substrate class from a gamma-hexachlorocyclohexane-degrading bacterium, Sphingomonas paucimobilis UT26

The linB gene product (LinB), which is involved in the degradation of gamma -hexachlorocyclohexane in Sphingomonas paucimobilis UT26, is a member of ha loalkane dehalogenases with a broad range of substrate specificity. Elucida tion of the factors determining its substrate specificity is of interest. A iming to facilitate purification of recombinant LinB protein for site-direc ted mutagenesis analysis, a 6-histidyl tail was added to the C-terminus of LinB. The His-tagged LinB was specifically bound with Ni-NTA resin in the b uffer containing 10 mM imidazole. After elution with 500 mM imidazole, quan titative recovery of protein occurred. The steady-state kinetic parameters of the His-tagged LinB for four substrates were in good agreement with that of wild-type recombinant LinB. Although the His-tagged LinB expressed in a n average of 80% of the activity of the wild type LinB for 10 different sub strates, the decrease was very similar for different substrates with the st andard deviation of 5.5%. The small activity reduction is independent of th e substrate shape, size, or number of substituents, indicating that the His -tagged LinB can be used for further mutagenesis studies. To confirm the su itability of this system for mutagenesis studies, two mutant proteins with substitution in putative halide binding residues (W109 and F151) were const ructed, purified, and tested for activity. As expected, complete loss in ac tivity of W109L and sustained activity of F151W were observed. (C) 1999 Aca demic Press.