In Vivo Reshaping the Catalytic Site of Nucleoside 2′-Deoxyribosyltransferase for Dideoxy- and Didehydronucleosides via a Single Amino Acid Substitution

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 3 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.Nucleoside 2Ј-deoxyribosyltransferases (NDTs 2 ; EC 2.4.2.6) catalyze the transfer of the deoxyribosyl group (dR) from one base (X) to another base (Y), in the following reaction: dX ϩ Y % dY ϩX (where dX and dY represent the corresponding 2Ј-deoxyribonucleosides). These enzymes initially found in Lactobacillus participate in nucleoside salvage (1). NDTs are also present 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 (5-7). The transferase reaction proceeds via a ping-pong bi-bi mechanism in which the first product is released leaving a covalent deoxyribosyl-enzyme intermediate before the second substrate combines with it (5, 8, 9, ). 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).Enzymatic synthesis catalyzed by NDTs (10, 14) has several advantages over chemical methods. Indeed, chemical glycosylation increases the difficulty of obtaining the correct regioselectivity generating few secondary products (15). Furthermore, the instability of some intermediates under the conditions used for synthetic manipulations may be problematic, as demonstrated by the acid lability of purine ...

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].…”

“…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.…”

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

“…LrNDT shows lower K m values for dUrd than for Cyt, according to the ping-pong mechanism described for other NDTs (15,16). It is notable that the k cat and k cat /K m values for both substrates are higher than those described for other NDTs (15,16,29,41).…”

“…This substrate specificity could be explained according to the catalytic active site structure (Fig. 6), where the presence of a 2Ј-OH or a 2Ј-fluor atom does not allow the interaction by hydrogen bonds to carboxylate the oxygen atom of the putative catalytic Glu101, whereas the 3Ј-OH of the sugar shows the normal hydrogen bond essential for catalysis (1,29). In this sense it is interesting that LrNTD shows a high sequence identity with other described NDTs from lactobacilli, where the essential amino acids involved in catalysis are conserved.…”

“…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).…”