ALTERATION OF THE SUBSTRATE RANGE OF HALOALKANE DEHALOGENASE BY SITE-DIRECTED MUTAGENESIS

We attempted to expand the range of chlorinated solvents degraded by X anthobacter autotrophicus GJ10 to include trichloroethylene by the rat ional modification of the enzyme haloalkane dehalogenase. The amino ac ids Phe164, Asp170, Phe172 and Trp175 were individually replaced with alanine by site-directed mutagenesis. All substitutions produced enzym es with lower than wild type activity with 1,2-dichloroethane. The Phe 164Ala and Asp170Ala mutants were 3 and 2 times more active than was t he wild type enzyme in dechlorinating 1,6-dichlorohexane. The Asp170Al a mutant resembled the wild type enzyme in its relative activity again st longer chain substrates. No mutant was active with trichloroethylen e. (C) 1998 John Wiley & Sons, Inc.