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.
Highlights
JULY 18, 2008 VOLUME 283 NUMBER 29 ent in some species of Streptococcus [2] and in parasitic unicellular eukaryotic organisms such as Crithidia luciliae [3] and Trypanosoma brucei [4]
The L. leichmannii ndt gene was used because the x-ray structure of Ll-NDT is known and the function of several amino acids involved in substrate binding and catalysis are defined by sitedirected mutagenesis experiments [21, 33]
L. fermentum ndt gene was mutagenized, because Lf-NDT is quite distant from Ll-NDT (33% identity, data not shown), whereas the amino acids important for the transfer of the 2Ј-deoxyribose are conserved
Summary
JULY 18, 2008 VOLUME 283 NUMBER 29 ent in some species of Streptococcus [2] and in parasitic unicellular eukaryotic organisms such as Crithidia luciliae [3] and Trypanosoma brucei [4]. Two different enzymes have been described in Lactobacillus helveticus and Lactobacillus leichmannii; they are NDT I, specific for purines, and NDT II, which transfers dR between purine-purine, pyrimidine-pyrimidine, and purine-pyrimidine [5,6,7]. The stereospecificity of the reaction (only the  anomer is formed) and the tolerance of NDT II for structural variations in the acceptor base have been exploited to synthesize various nucleoside analogs (10 –14). Because several nucleoside analogs acting as antiviral or anticancer agents have modifications on their sugar moiety and despite the number of reported synthetic methods, the development of alternative approaches is still relevant. We address the question of sugar tolerance of NDT at the 3Ј position by selecting enzymes with improved 2,3dideoxyribosyl (ddR) transfer activity between various bases. A single amino acid substitution, A15T in Lf-NDT or G9S in Ll-NDT, allowed the conversion of JOURNAL OF BIOLOGICAL CHEMISTRY 20053
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