THE ROLE OF SPONTANEOUS CAP DOMAIN MUTATIONS IN HALOALKANE DEHALOGENASE SPECIFICITY AND EVOLUTION

The first step in the utilization of the xenobiotic chlorinated hydroc arbon 1,2-dichloroethane by Xanthobacter autotrophicus is catalyzed by haloalkane dehalogenase (DhlA). The enzyme hydrolyses 1-haloalkanes t o the corresponding alcohols. This allows the organism to grow also on short-chain (C-2-C-4) 1-chloro-n-alkanes. We have expressed DhlA in a strain of Pseudomonas that grows on long-chain alcohols and have sele cted 12 independent mutants that utilize 1-chlorohexane. Six different mutant enzymes with improved K-m or V-max values with 1-chlorohexane were obtained. The sequences of the mutated dhlA genes showed that sev eral mutants had the same 11-amino acid deletion, two mutants carried a different point mutation, and three mutants had different tandem rep eats. All mutations occurred in a region encoding the N-terminal part of the cap domain of DhlA, and it is concluded that this part of the p rotein is involved in the evolution of activity toward xenobiotic subs trates.