In vivo reshaping the catalytic site of nucleoside 2'-deoxyribosyltransferase for dideoxy- and didehydronucleosides via a single amino acid substitution.
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.
Kaminski
Pierre Alexandre
PA
Institut Pasteur, Unité de Chimie Organique, CNRS, URA2128, Paris Cedex 15, France. akaminsk@pasteur.fr
Dacher
Priscilla
P
Dugué
Laurence
L
Pochet
Sylvie
S
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D020134:Catalytic Domain; D006859:Hydrogen / Q000737:chemistry*; D007700:Kinetics; D052197:Limosilactobacillus fermentum / Q000235:genetics / Q000378:metabolism; D052199:Lactobacillus leichmannii / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009705:Nucleosides / Q000737:chemistry* / Q000378:metabolism*; D010100:Oxygen / Q000737:chemistry*; D010430:Pentosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013379:Substrate Specificity
D009705:Nucleosides; D006859:Hydrogen; D010430:Pentosyltransferases; C021888:nucleoside deoxyribosyltransferase; D010100:Oxygen