Lactobacillus reuteri 2′-Deoxyribosyltransferase, a Novel Biocatalyst for Tailoring of Nucleosides

Abstract

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% ␣-helix, 10% ␤-strand, 16% ␤-sheet, and 19% random coil. LrNDT was thermostable with a melting temperature (T m ) of 64°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°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.Nucleoside 2Ј-deoxyribosyltransferases (NDTs) (EC 2.4.2.6) catalyze the exchange between the purine or pyrimidine base of 2Ј-deoxyribonucleosides and free pyrimidine or purine bases (10, 25). These enzymes are specific for 2Ј-deoxyribonucleosides, regioselective (N-1 glycosylation in pyrimidine and N-9 in purine), and stereoselective (␤-anomers are exclusively formed) (26) (Fig. 1).Deoxyribosyltransferases are classified into two classes depending on their substrate specificity: type I (NDT I), specific for purines (Pur 7 Pur), and type II (NDT II), which catalyzes the transfer between purines and/or pyrimidines (Pur 7 Pur, Pur 7 Pyr, Pyr 7 Pyr) (10, 25). These enzymes were initially described for lactobacilli (27,28), and they are involved in the nucleoside salvage pathway for DNA synthesis (23), although this remains unclear in Lactococcus lactis subsp. lactis (36). NDTs have been also found in some species of Streptococcus (11), in parasitic unicellular eukaryotic organisms such as Crithidia luciliae (49, 50), in Trypanosoma brucei (6), and in Borrelia burgdorferi (33). NDTs from Lactobacillus helveticus and Lactobacillus leichmannii have been well studied (2,25,26,28,29), and their kinetic mechanisms as well as their catalytic and substrate binding sites have been characterized. The transferase reaction proceeds via a ping-pong bi-bi mechanism by formation of a covalent deoxyribosyl enzyme intermediate (3,15,16). Likewise, a glutamyl residue (Glu98) has been proven essential for activity (40,41,46). Enzymatic natural and nonnatural nucleoside synthesis in a one-pot reaction by NDTs provides an interesting alternative to traditional multistep chemical methods (13,34). Indeed, chemical glycosylation includes several protection-deprotection steps and the use of chemical reagents and organic solvents that are expensive and environmentally harmful. Whereas previously described NDTs accept different nucleosides fr...

2′-Deoxyribosyltransferase from Bacillus psychrosaccharolyticus: A Mesophilic-Like Biocatalyst for the Synthesis of Modified Nucleosides from a Psychrotolerant Bacterium

“…In this case, BpNDT also behaves as a mesophilic enzyme since the kinetic parameters we determined using dCyd and Ade as nucleoside donor and base acceptor, respectively (dCyd: K M = 2.0 ± 0.3 mM and k cat (s −1 ) = 24.6 ± 0.1; Ado: K M = 0.4 ± 0.1 mM and k cat (s −1 ) = 28.2 ± 0.1) compare well with published values for other mesophilic homologues [18,27,41,42].…”

“…This substrate specificity is similar to other well-known NDTs [12,18,19] except for the strong preference for Hyp as acceptor, only comparable with that of L. lactis subsp. lactis NDT [26].…”

“…Indeed, although we have observed that some loops flanking the substrate cavity of BpNDT are more flexible than in the mesophilic LlNDT or LhPDT (Figures 4 and 5), it is obvious from the thermal inactivation experiments that this structural feature does not result in a heat-labile enzyme: BpNDT exhibits highest activity at 40 • C and pH 8 [25]. In fact, it is active under these conditions for a longer period of time than the mesophilic LrNDT: the half-time at 60 • C of BpNDT (21.8 h) is remarkably higher than the one observed for LrNDT (17.9 min) [18].…”

“…In turn, the most and least preferred acceptors are hypoxanthine (Hyp) and uracil (Ura), respectively. Compared to previously described NDTs [18,19,26,27], BpNDT exhibits higher specific activities, except when compared to LrNDT. The synthesis of dIno from Hyp and dAdo proceeds 5.4 times faster in the presence of BpNDT than with LrNDT or PDT from Lactobacillus helveticus (LhPDT), whereas the transfer between Thd and Ura is twice faster with LrNDT or Lactococcus lactis NDT (LlcNDT) than with BpNDT.…”

“…BpNDT was able to catalyze the transfer reaction using most of non-natural bases as acceptors, with a slight decrease in yield with respect to the one described for LrNDT [18] when the donor is dUrd. As observed in the case of natural bases, higher activities were obtained when purine bases were used, such as benzimidazole, 2,6-diaminopurine and 2-fluoroadenine.…”