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In Vivo Reshaping the Catalytic Site of Nucleoside 2'-Deoxyribosyltransferase for Dideoxy- and Didehydronucleosides via a Single Amino Acid Substitution.

Abstract : Nucleoside 2'-deoxyribosyltransferases catalyze the transfer of 2-deoxyribose between bases and have been widely used as biocatalysts to synthesize a variety of nucleoside analogs. The genes encoding nucleoside 2'-deoxyribosyltransferase (ndt) from Lactobacillus leichmannii and Lactobacillus fermentum underwent random mutagenesis to select variants specialized for the synthesis of 2',3'-dideoxynucleosides. An Escherichia coli strain, auxotrophic for uracil and unable to use 2',3'-dideoxyuridine, cytosine, and 2',3'-dideoxycytidine as a source of uracil was constructed. Randomly mutated lactobacilli ndt libraries from two species, L. leichmannii and L. fermentum, were screened for the production of uracil with 2',3'-dideoxyuridine as a source of uracil. Several mutants suitable for the synthesis of 2',3'-dideoxynucleosides were isolated. The nucleotide sequence of the corresponding genes revealed a single mutation (G --> A transition) leading to the substitution of a small aliphatic amino acid by a nucleophilic one, A15T (L. fermentum) or G9S (L. leichmannii), respectively. We concluded that the "adaptation" of the nucleoside 2'-deoxyribosyltransferase activity to 2,3-dideoxyribosyl transfer requires an additional hydroxyl group on a key amino acid side chain of the protein to overcome the absence of such a group in the corresponding substrate. The evolved proteins also display significantly improved nucleoside 2',3'-didehydro-2',3'-dideoxyribosyltransferase activity.
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Submitted on : Wednesday, July 16, 2008 - 5:33:01 PM
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Pierre-Alexandre Kaminski, Priscilla Dacher, Laurence Dugué, Sylvie Pochet. In Vivo Reshaping the Catalytic Site of Nucleoside 2'-Deoxyribosyltransferase for Dideoxy- and Didehydronucleosides via a Single Amino Acid Substitution.. Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2008, 283 (29), pp.20053-20059. ⟨10.1074/jbc.M802706200⟩. ⟨pasteur-00298608⟩

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