Substrate specificity of the purine-2′-deoxyribonucleosidase of <i>Crithidia luciliae</i>

Steenkamp, Daniël J.; Hälbich, T J F

doi:10.1042/bj2870125

Cited by 14 publications

(5 citation statements)

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“…This value is similar to the molecular mass calculated from the amino acid sequence (109 kDa). These results agree with previous reports for this kind of enzyme (1,3,14,25,28,36,49,50). In order to characterize LrNDT, several properties have been further investigated.…”

Section: Purification and Structural Characterization Of Type II Nuclsupporting

confidence: 82%

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

Fernández‐Lucas

Acebal

Sinisterra³

et al. 2010

Appl Environ Microbiol

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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...

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Section: Purification and Structural Characterization Of Type II Nuclsupporting

confidence: 82%

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

Fernández‐Lucas

Acebal

Sinisterra³

et al. 2010

Appl Environ Microbiol

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“…Whether they contain the two types of activities remains unknown for the moment. A purine trans-N-deoxyribosyltransferase similar to DRTase I was purified from the protozoan parasite Crithidia luciliae (32,33). Because this parasite does not synthesize purines de novo and is deficient in adenosine deaminase, it was postulated that the deoxyribosyltransferase may regulate the pool of nucleotides.…”

Section: Discussionmentioning

confidence: 99%

Functional Cloning, Heterologous Expression, and Purification of Two Different N-Deoxyribosyltransferases fromLactobacillus helveticus

Kaminski

2002

Journal of Biological Chemistry

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Lactobacillus helveticus contains two types of N-deoxyribosyltransferases: DRTase I catalyzes the transfer of 2-deoxyribose between purine bases exclusively whereas DRTase II is able to transfer the 2-deoxyribose between two pyrimidine or between pyrimidine and purine bases. An Escherichia coli strain, auxotrophic for guanine and unable to use deoxyguanosine as source of guanine, was constructed to clone the corresponding genes. By screening a genomic bank for the production of guanine, the L. helveticus ptd and ntd genes coding for DRTase I and II, respectively, were isolated. Although the two genes have no sequence similarity, the two deduced polypeptides display 25.6% identity, with most of the residues involved in substrate binding and the active site nucleophile Glu-98 being conserved. Overexpression and purification of the two proteins shows that DRTase I is specific for purines with a preference for deoxyinosine (dI) > deoxyadenosine > deoxyguanosine as donor substrates whereas DRTase II has a strong preference for pyrimidines as donor substrates and purines as base acceptors. Purine analogues were substrates as acceptor bases for both enzymes. Comparison of DRTase I and DRTase II activities with dI as donor or hypoxanthine as acceptor and colocalization of the ptd and add genes suggest a specific role for DRTase I in the metabolism of dI.

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“…ent in some species of Streptococcus (2) and in parasitic unicellular eukaryotic organisms such as Crithidia luciliae (3) and Trypanosoma brucei (4).…”

mentioning

confidence: 99%

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

Kaminski

Dacher

Dugué

et al. 2008

Journal of Biological Chemistry

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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 ...

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Substrate specificity of the purine-2′-deoxyribonucleosidase of Crithidia luciliae

Cited by 14 publications

References 20 publications

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

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

Functional Cloning, Heterologous Expression, and Purification of Two Different N-Deoxyribosyltransferases fromLactobacillus helveticus

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

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