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In vivo reshaping the catalytic site of nucleoside 2'-deoxyribosyltransferase for dideoxy- and didehydronucleosides via a single amino acid substitution.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2008 Jul 18; Vol. 283 (29), pp. 20053-9. Date of Electronic Publication: 2008 May 16. - Publication Year :
- 2008
-
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.
- Subjects :
- Amino Acid Sequence
Amino Acid Substitution
Catalytic Domain
Kinetics
Limosilactobacillus fermentum genetics
Limosilactobacillus fermentum metabolism
Lactobacillus leichmannii genetics
Lactobacillus leichmannii metabolism
Models, Molecular
Molecular Sequence Data
Mutation genetics
Pentosyltransferases chemistry
Pentosyltransferases genetics
Protein Engineering
Protein Structure, Tertiary
Sequence Alignment
Substrate Specificity
Hydrogen chemistry
Nucleosides chemistry
Nucleosides metabolism
Oxygen chemistry
Pentosyltransferases metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0021-9258
- Volume :
- 283
- Issue :
- 29
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 18487606
- Full Text :
- https://doi.org/10.1074/jbc.M802706200