Abstract
A large number of nucleoside analogues and 2'-deoxynucleoside triphosphates (dNTP) have been synthesized to interfere with DNA metabolism. However, in vivo the concentration and phosphorylation of these analogues are key limiting factors. In this context, we designed enzymes to switch nucleobases attached to a deoxyribose monophosphate. Active chimeras were made from two distantly related enzymes: a nucleoside deoxyribosyltransferase from lactobacilli and a 5'-monophosphate-2'-deoxyribonucleoside hydrolase from rat. Then their unprecedented activity was further extended to deoxyribose triphosphate, and in vitro biosyntheses could be successfully performed with several base analogues. These new enzymes provide new tools to synthesize dNTP analogues and to deliver them into cells.
Highlights
The nucleoside deoxyribosyltransferase family contains hydrolases and transferases with different substrate specificities
Comparison of the structures and catalytic mechanisms of members of the nucleoside deoxyribosyltransferase family allows the design of unprecedented enzymes
NDT was chosen as a starting template because its transferase activity was more interesting from the synthetic point of view because it exchanges deoxyribose between pyrimidines and purines and, vice versa, between two pyrimidines and to a lesser extent between two purines
Summary
The nucleoside deoxyribosyltransferase family contains hydrolases and transferases with different substrate specificities. Active chimeras were made from two distantly related enzymes: a nucleoside deoxyribosyltransferase from lactobacilli and a 5-monophosphate-2-deoxyribonucleoside hydrolase from rat Their unprecedented activity was further extended to deoxyribose triphosphate, and in vitro biosyntheses could be successfully performed with several base analogues. In addition to the four canonical dNTPs, four noncanonical dNTPs bearing an exotic pyrimidine and one bearing an exotic purine are condensed by bacterial viruses [1, 2] This shows that DNA can host modified nucleobases that could serve, for example, to extend the genetic code or to induce mutagenesis. The demand for purified deoxyribonucleotides is high, for example, for DNA synthesis (PCR or DNA microarrays) and reverse transcription in vitro in academic research and medical diagnosis The atomic coordinates and structure factors (code 4HX9) have been deposited in the Protein Data Bank (http://wwpdb.org/)
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