Lactobacillus reuteri 2'-deoxyribosyltransferase, a novel biocatalyst for tailoring of nucleosides.
A novel type II nucleoside 2'-deoxyribosyltransferase from Lactobacillus reuteri (LrNDT) has been cloned and overexpressed in Escherichia coli. The recombinant LrNDT has been structural and functionally characterized. Sedimentation equilibrium analysis revealed a homohexameric molecule of 114 kDa. Circular dichroism studies have showed a secondary structure containing 55% alpha-helix, 10% beta-strand, 16% beta-sheet, and 19% random coil. LrNDT was thermostable with a melting temperature (T(m)) of 64 degrees C determined by fluorescence, circular dichroism, and differential scanning calorimetric studies. The enzyme showed high activity in a broad pH range (4.6 to 7.9) and was also very stable between pH 4 and 7.9. The optimal temperature for activity was 40 degrees C. The recombinant LrNDT was able to synthesize natural and nonnatural nucleoside analogues, improving activities described in the literature, and remarkably, exhibited unexpected new arabinosyltransferase activity, which had not been described so far in this kind of enzyme. Furthermore, synthesis of new arabinonucleosides and 2'-fluorodeoxyribonucleosides was carried out.
Fernández-Lucas
Jesús
J
Department of Biochemistry and Molecular Biology I, Faculty of Biology, Complutense University of Madrid, C/ José Antonio Nováis 2, 28040 Madrid, Spain.
Acebal
Carmen
C
Sinisterra
José V
JV
Arroyo
Miguel
M
de la Mata
Isabel
I
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004798:Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D052200:Limosilactobacillus reuteri / Q000201:enzymology* / Q000235:genetics; D008970:Molecular Weight; D009705:Nucleosides / Q000378:metabolism*; D010430:Pentosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization; D017433:Protein Structure, Secondary
D001426:Bacterial Proteins; D004798:Enzymes; D009705:Nucleosides; D010430:Pentosyltransferases; C101967:arabinosyltransferase; C420226:nucleoside deoxyribosyltransferase II