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.
Department of Biochemistry, University of Groningen, The Netherlands.
F
F
Pries
A J
AJ
van den Wijngaard
R
R
Bos
M
M
Pentenga
D B
DB
Janssen
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