Abstract
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.
Highlights
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The sequences of themutated dhlA genes alkanes and a,o-dichloro-n-alkanes, but there is hardly any showed that several mutants had the same 11-amino activity with 1-chlorohexane or chloroalkanes of longer chain acid deletion, two mutants carried a different mpoui-nt length [1].The enzyme has some activity with long-chain brotation, and three mutants had different tandem repeamtso.alkanes (C5-Clo),suggesting that the active site canaccom
Our results present experimental eviof long chain chloroalkanes
Summary
THE ROLE OF SPONTANEOUS CAP DOMAIN MUTATIONS IN HALOALKANE DEHALOGENASE SPECIFICITY AND EVOLUTION Pries, Frens; Wijngaard, Arjan J. van den; Bos, Rolf; Pentenga, Marjan; Janssen, Dick B. The sequences and activity of the mutant enzymes that were obtained indicate that short direct repienats the N-terminal paorftthe cap domain play an important role in the evolution of haloalkane dehalogenasespecificity. This indicates that the covalentalkyl-enzyme intermediate, formed by nucleophilic displacement of the halogen by Asp124is, hydrolyzed by nucleophilic attack of a water molecule on the carbonyl carbon atom.His289probably activates this water molecule by subtracting a proton, with assistanceofAspZ6’(8).Two achievewdith the recombinabnat cterial strain Pseudomonas GJ31(pPJ20).Pseudomonas GJ31 is capable of growing on long-chain alcohols. 33 bp(Pdhelee"t-iAonla"') 9 bp tandedmuplicatio(Pnhe172-Ala174) 6 bp tandedmuplicatio(nMet15Z-Thr153) 3bta0pndedmuplicatio(1n1e145-Asp154)
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