The role of spontaneous cap domain mutations in haloalkane dehalogenase specificity and evolution.
The first step in the utilization of the xenobiotic chlorinated hydrocarbon 1,2-dichloroethane by Xanthobacter autotrophicus is catalyzed by haloalkane dehalogenase (Dh1A). The enzyme hydrolyses 1-haloalkanes to the corresponding alcohols. This allows the organism to grow also on short-chain (C2-C4) 1-chloro-n-alkanes. We have expressed Dh1A in a strain of Pseudomonas that grows on long-chain alcohols and have selected 12 independent mutants that utilize 1-chlorohexane. Six different mutant enzymes with improved Km or Vmax values with 1-chlorohexane were obtained. The sequences of the mutated dh1A genes showed that several mutants had the same 11-amino acid deletion, two mutants carried a different point mutation, and three mutants had different tandem repeats. All mutations occurred in a region encoding the N-terminal part of the cap domain of Dh1A, and it is concluded that this part of the protein is involved in the evolution of activity toward xenobiotic substrates.
Pries
F
F
Department of Biochemistry, University of Groningen, The Netherlands.
van den Wijngaard
A J
AJ
Bos
R
R
Pentenga
M
M
Janssen
D B
DB
D000595:Amino Acid Sequence; D001483:Base Sequence; D005075:Biological Evolution; D003352:Corynebacterium / Q000201:enzymology; D004269:DNA, Bacterial; D006586:Hexanes / Q000378:metabolism; D006843:Hydrocarbons, Chlorinated / Q000378:metabolism; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D011549:Pseudomonas; D013379:Substrate Specificity
D004269:DNA, Bacterial; D006586:Hexanes; D006843:Hydrocarbons, Chlorinated; D006867:Hydrolases; C046137:haloalkane dehalogenase; C053283:1-chlorohexane