Nucleoside Deoxyribosyltransferase Research Articles

Studies on enzymatic transglycosylation catalyzed by bacterial Nucleoside deoxyribosyltransferase II and Nucleoside phosphorylase for the synthesis of pyrimidine 2′-Deoxyribonucleosides containing modified heterocyclic base

A comparative analysis of enzymatic transglycosylation reactions catalyzed by Lactobacillus leichmannii NDT II and Escherichia coli PNP in the presence of various glycosyl donors and under different conditions was performed to synthesize modified 2ʹ-deoxynucleosides. Substitution of Escherichia coli PNP with Lactobacillus leichmannii NDT II allowed the synthesis of various deoxynucleosides from different glycosyl donors in 39–100% yields. The replacement of thymidine as a commonly used NDT substrate with 7-Methyl-2′-deoxyguanosine as a novel glycosyl-donor allowed the enzymatic synthesis of 2′-deoxycytidine derivatives and 5-substituted 2′-deoxyuridine derivatives, which is essential for further elaboration of eco-friendly technologies for synthesis of base-modified nucleosides.

Isolation and Characterization of Engineered Nucleoside Deoxyribosyltransferase with Enhanced Activity Toward 2'-Fluoro-2'-Deoxynucleoside.

Nucleoside deoxyribosyltransferase (NDT) is an enzyme that replaces the purine or pyrimidine base of 2'-deoxyribonucleoside. This enzyme is generally used in the nucleotide salvage pathway in vivo and synthesizes many nucleoside analogs in vitro for various biotechnological purposes. Since NDT is known to exhibit relatively low reactivity toward nucleoside analogs such as 2'-fluoro-2'-deoxynucleoside, it is necessary to develop an enhanced NDT mutant enzyme suitable for nucleoside analogs. In this study, molecular evolution strategy via error-prone PCR was performed with ndt gene derived from Lactobacillus leichmannii as a template to obtain an engineered NDT with higher substrate specificity to 2FDU (2'-fluoro-2'-deoxyuridine). A mutant library of 214 ndt genes with different sequences was obtained and performed for the conversion of 2FDU to 2FDA (2'-fluoro-2'-deoxyadenosine). The E. coli containing a mutant NDT, named NDTL59Q, showed 1.7-fold (at 40°C) and 4.4-fold (at 50°C) higher 2FDU-to-2FDA conversions compared to the NDTWT, respectively. Subsequently, both NDTWT and NDTL59Q enzymes were over-expressed and purified using a His-tag system in E. coli. Characterization and enzyme kinetics revealed that the NDTL59Q mutant enzyme containing a single point mutation of leucine to glutamine at the 59th position exhibited superior thermal stability with enhanced substrate specificity to 2FDU.

Bioinformatics of thymidine metabolism in Trypanosoma evansi: exploring nucleoside deoxyribosyltransferase (NDRT) as a drug target.

Trypanosoma evansi, the causative agent of surra or camel trypanosomiasis, is characterized by the widest geographic distribution and host range among the known trypanosomes. Its zoonotic importance and increasing evidence of drug resistance necessitate the discovery of new drug targets. The drug discovery process entails finding an exploitable difference between the host and the parasite. In this study, the thymidine metabolic pathways in camel and T. evansi were compared by analyzing their metabolic maps, protein sequences, domain and motif contents, phylogenetic relationships, and 3D structure models. The two organisms were revealed to recycle thymidine differently: performed by thymidine phosphorylase in camels (Camelus genus), this role in T. evansi was associated with nucleoside deoxyribosyltransferase (NDRT), a unique trypanosomal enzyme absent in camels. Thymidine in T. evansi seems to be governed by thymine through NDRT, whereas in camels, thymidine can be produced from thymidylate via 5'-nucleotidase. As a result, NDRT may be a promising drug target against T. evansi.

Establishment of a high throughput-screening system for nucleoside deoxyribosyltransferase II mutant enzymes with altered substrate specificity

Nucleoside deoxyribosyltransferase II (NDT) catalyzes the transglycosylation reaction of the 2'-deoxyribose moiety between purine and/or pyrimidine bases and has been widely used in the synthesis of nucleoside analogs. The high specificity of NDT for 2'-deoxyribose limits its applications. Because 2'C- and/or 3'C-modified nucleosides have been widely used as antiviral or antitumour agents, improving the activity of NDT towards these modified nucleosides by protein engineering is an area of interest to the pharmaceutical industry. NDT engineering is hindered by a lack of effective screening methods. This study developed a high-throughput screening system, which was established by nucleoside deoxyribosyltransferase II-cytidine deaminase co-expression, indophenol colorimetric assay and whole-cell catalysis. A high-throughput screening system for NDT was established for the first time. This system can be applied to detect NDT-specific activity for a variety of cytidine analogs with glycosyl and base modifications, such as 5-aza-2'-deoxycytidine, 2',3'-dideoxycytidine, cytosine-β-d-arabinofuranoside. In this study, we adopted the semi-rational design of NDT and constructed a mutant library of NDT from Lactobacillus helveticus (LhNDT) by site-saturation mutagenesis. Over 600 mutants were screened, and a variant with up to a 5.2-fold higher conversion rate of 2',3'-dideoxyinosine was obtained.

Phosphodeoxyribosyltransferases, Designed Enzymes for Deoxyribonucleotides Synthesis

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.

Biotechnology of Nucleic Acid Constituents - State of the Art and Perspectives

A survey of the chemo-enzymatic methodology for the preparation of nucleosides of biological importance is presented. The emphasis is made on the enzymatic transglycosylation and glycosylation employing the nucleoside phosphorylases and N-deoxyribosyltransferases. Application of the whole cells as biocatalysts as well as purified enzymes from wild-type bacteria and recombinant enzymes is considered. Keywords: N-Deoxyribosyltransferases, nucleoside deoxyribosyltransferase, Nucleoside Phosphorylases, Escherichia coli, cytidine deaminase (CDase)

Evidence for dual effect of bile acids on thymidine anabolism and catabolism by the regenerating rat liver

Bile acids have been reported to modify DNA synthesis by rodent livers in regeneration, which may be due in part to their ability to interact with the machinery responsible for deoxyribonucleotide synthesis. The aim of this work was to gain information on the effect of taurocholate (TC) on both anabolic and catabolic pathways accounting for the fate of [methyl-14C]thymidine in the liver of two-third hepatectomized rats. Using high-pressure liquid chromatography, the soluble fraction of liver homogenate was used to measure the ability of TC to modify both the rate of thymidine monophosphate formation from thymidine — i.e., thymidine kinase (TK) activity — and the rate of thymidine release from thymidine, which is the result of at least three different reactions catalyzed by thymidine phosphorylase, nucleosidase and nucleoside deoxyribosyl transferase. TC was found to induce a dose-dependent inhibition of both processes. The nature of this inhibition seems to be in part competitive. Apparent Ki value were 1.5 mM for TK and 4 mM for thymidine release. These inhibitory effects were mimicked by glycocholate but not by taurine. To investigate the relevance of the TC-induced modification of anabolism and catabolism in the whole organ, experiments on regenerating perfused rat livers were carried out. The donors underwent two-third hepatectomy 24 h before liver isolation. They were either fasted during this period (F) or allowed free access to food (NF). DNA synthesis, as measured by [methyl-14C]thymidine incorporation into DNA, was significantly increased in both groups, as compared with control non-hepatectomized animals. However, enhancement in DNA synthesis in group F was only 50% of the value found in the NF group. Intravenous TC administration before and/or during liver perfusions induced a dose-dependent recovery of DNA synthesis in the F group. This effect was accompanied by opposed modifications in the amount of radiolabelled metabolites contained in the non-DNA fraction of liver homogenate, consistent with a marked inhibition of thymidine catabolism. These results suggest that, in addition to the previously reported effects of TC on thymidine anabolism, bile acids are also able to affect thymidine catabolism. The overall results of this dual effect on the fate of thymidine in the regenerating rat liver depend on the metabolic situation. Under circumstances of no nutrient restriction, the effect of TC is characterized by inhibition of thymidine incorporation into DNA. By contrast, under depressed DNA synthesis due to fasting, the overall effect of TC is a partial recovery of this process.

Nucleoside deoxyribosyltransferase and inosine phosphorylase activity in lactic acid bacteria

Phosphate-independent nucleoside deoxyribosyltransferase activity was detected in Aerococcus viridans, Leuconostoc mesenteroides, two species of Pediococcus, eight obligately homofermentative and three obligately heterofermentative lactobacilli. In cellfree extracts of one strain of Lactococcus, one strain of Streptococcus and three strains of facultatively heterofermentative lactobacilli no phosphate-independent nucleoside deoxyribosyltransferase could be detected and inosine nucleoside phosphorylase was the only enzyme catalysing pentosyl transfer to purines. A classification based on the presence or absence of these enzymes separates Streptococcus and Lactococcus from the remaining genera which is in agreement with studies using anti-aldolase sera and 16S rRNA oligonucleotide catalogues.

Crystallization and preliminary X-ray investigation of recombinant Lactobacillus leichmannii nucleoside deoxyribosyltransferase.

Crystals of recombinant bacterial nucleoside deoxyribosyltransferase have been grown from solutions of ammonium sulfate. The crystals are cubic, space group I23 or I2(1)3; the axial length is 151.1(2) A. The crystals are stable to x-rays for at least 5 days and diffract beyond 2.8-A resolution. It appears that the molecule, which is a hexamer, utilizes the symmetry of the space group, resulting in two or three subunits per asymmetric unit.

Formation of 3-(2′-deoxyribofuranosyl) and 9-(2′-deoxyribofuranosyl) nucleosides of 8-substituted purines by nucleoside deoxyribosyltransferase

Earlier results suggested that although the N-deoxyribosyltransferase from lactobacilli is a convenient tool for the preparation of analogs of 2′-deoxyadenosine, 8substituted purines do not act as substrates. However, eight of nine 8-substituted purines that were examined proved to be substrates for the transferase from Lactobacillus leichmannii, and deoxyribonucleosides of four of these bases have been prepared. The substituents at C-8 of the purine greatly affect the rate of deoxyribosyl transfer to the base, and in all cases the rate is slower than transfer to purines lacking an 8-substituent. The 8-substituent also affects the nature of the nucleoside formed. With the electron-donating methyl group at position 8 of adenine, the transferase forms the expected 8-methyl-9-(2′-deoxyribofuranosyl)adenine. However, when purines bearing an electron-withdrawing substituent at the 8-position are used as substrates, the deoxyribosyl moiety is preferentially transferred to N-3 of the base. In the case of 8trifluoromethyladenine the 3-deoxyribonucleoside is the only product detectable. With 8-bromo or 8-chloroadenine as substrate the 3 and 9-deoxyribonucleosides can both be isolated from the enzymatic reaction mixture. Time course studies indicated that with thymidine and 8-bromoadenine as substrates the 3-deoxyribonucleoside is initially the major product, but that the 9-deoxyribonucleoside becomes the major product after long incubation periods. Negligible interconversion of these nucleosides occurs in the absence of transferase, but conversion in either direction occurs readily in the presence of the enzyme. Significant hydrolysis of pyrimidine and purine deoxyribonucleosides occurs in the presence of the transferase. This was more obvious during the course of reactions involving 8-substituted purines because the slowness of deoxyribosyl transfer required longer incubation periods and larger amounts of enzyme. The hydrolysis is proportional to enzyme concentration, little affected by the nature of the base and is attributed to hydrolysis of a deoxyribosyl derivative of the transferase which is an obligatory intermediate of deoxyribosyl transfer. 8-Trifluoromethyl-3-(2′-deoxyribofuranosyl) adenine, 8-methyl-9-(2′-deoxyribofuranosyl)adenine, and 8-bromo-9-(2′-deoxyribofuranosyl) adenine were tested for their ability to inhibit the growth of CCRFCEM cells in culture. Unlike the potent 2-halogeno-2′-deoxyadenosine derivatives, these three nucleosides cause less than 50% inhibition at concentrations up to 100 μ m.

Analogs of 2′-deoxyadenosine: Facile enzymatic preparation and growth inhibitory effects on human cell lines

Nucleoside deoxyribosyltransferase has been extensively purified from extracts of Lactobacillus leichmannii. The enzyme was initially co-purified with ribonucleotide reductase and then separated from the latter by affinity chromatography on dGTP-Sepharose. A new procedure is described for the synthesis of 6-( p-aminobenzylamino)purine. The latter was diazotized and reacted with Sepharose substituted with m-phenylenediamine to produce an affinity material for chromatography of the transferase. Some properties of the purified transferase are described, including remarkable stability to heat and to guanidine hydrochloride. With thymidine as donor, the transferase had a very broad specificity for the purine acceptor, the only modifications not tolerated being the loss of the tautomeric N in the imidazole ring and, possibly, substitution at the 8-position. The enzyme was used for the convenient synthesis of twelve analogs of 2'-deoxyadenosine on the 100–400 mg scale with an average yield of 64 per cent. The method was readily applicable to larger scale preparations and the enzyme was recoverable from the final reaction mixtures in good yield. Of the nucleosides synthesized, the 2-chloro and 2-bromo analogs of 2'-deoxyadenosine were especially good inhibitors of the growth of KB and CCRF-CEM cells in culture.

Nucleoside deoxyribosyltransferase-II from Lactobacillus helveticus. Substrate specificity studies. Pyrimidine bases as acceptors

The nucleoside deoxyribosyltransferase (nucleoside:purine (pyrimidine) deoxyribosyltransferase, EC 2.4.2.6) fraction catalyzing specifically the transfer of the deoxyribosyl moiety from a purine (or a pyrimidine) to a pyrimidine (or a purine) exhibits a broad specificity for the acceptor base. With a pyrimidine base as the acceptor a -OH or -SH group adjacent to the N-1 atom is essential. A substituent on position 6 hinders the reaction. On positions 4 and 5 various substituents were found to influence the reaction rate and some of them give non-competent substrates. A few anomalous cases are also discussed in relation with the role of N-3. Deoxyribonucleosides can also be obtained with non-pyrimidine rings.

60] Nucleoside deoxyribosyltransferase from Lactobacillus helveticus

Publisher Summary This chapter describes the enzyme nucleoside deoxyribosyltransferase from Lactobacillus helveticus. Nucleoside deoxydbosyltransferase (originally named trans-N-glycosidase 13, then trans-N-deoxyribosylase), catalyzes three types of transfer of the deoxyribosyl moiety according to the nature of the donor and acceptor bases. Lactobacillus helveticus extracts contain two different enzymes: nucleoside deoxyribosyltransferase-I or purine nucleoside and nucleoside deoxyribosyltransferase-II or purine (pyrimidine) nucleoside. Because the amount of these two types of enzyme varies significantly from strain to strain, it is desirable to perform the assay reactions. Hypoxanthine formed (using deoxyinosine as donor) is rapidly oxidized to uric acid by xanthine oxidase. The activity is measured at 40°. The change in absorbance is followed at 290 nm where most common bases and nucleosides have low extinction coefficients. It is important to check that the rate of the oxidation of hypoxanthine is not limiting and that no deoxyinosine hydrolase, adenine deaminase, or nucleoside phosphorylase are present.

Search for thymidine phosphorylase, nucleoside deoxyribosyltransferase and thymidine kinase in Moraxella, Acinetobacter, and allied bacteria.

Eighteen strains of Gram‐negative, rodshaped bacteria, including Moraxella kingii, “New Moraxella” Bijsterveld, “New Moraxella” Sutton et al., M. nonliquefaciens, M. lacunata, M. bovis, M. osloensis, M. phenylpyrouvica, M. atlantae (new species to be described), M. urethralis (tentative designation) and Acinetobacter, were examined for enzyme activities corresponding to thymidine phosphorylase, nucleoside deoxyribosyltransferase and thymidine kinase. The two strains of M. kingii examined as well as “New Moraxella” Bijsterveld and “New Moraxella” Sutton et al., revealed activities corresponding to the three enzymes. One of two M. nonliquefaciens strains, the type strain, was found to contain a thymidine kinase of low activity, but had no activities corresponding to the enzymes thymidine phosphorylase or nucleoside deoxyribosyltransferase. Significant activities of any of the three enzymes were not detected in the other strains examined.

Search for thymidine phosphorylase, nucleoside deoxyribosyltransferase and thymidine kinase in genus Neisseria.

The enzymes thymidine phosphorylase, nucleoside deoxyribosyltransferase and thymidine kinase were searched for in species of Neisseria. Activities corresponding to these enzymes could not be found in the species examined.

Separation of nucleoside deoxyribosyltransferase into four active components by affinity chromatography.

Separation of nucleoside deoxyribosyltransferase into four active components by affinity chromatography.

Utilization of thymine, thymidine and TMP by Neisseria meningitidis. 2. Lack of enzymes for specific incorporation of exogenous thymine, thymidine and TMP into DNA.

Activities corresponding to thymidine phosphorylase and nucleoside deoxyribosyl transferase could not be found in Neisseria meningitidis. 14C‐2‐thymine was bound to or taken up by whole cells, but this did not result in labelling of the TMP, TDP or TTP pools. No significant thymidine kinase activity could be induced in N. meningitidis. 14C‐2‐thymidine which was bound to or taken up by whole cells resulted in faint labelling of the TMP, TDP and TTP pools after prolonged incubation, but no radioactive thymine appeared. The N. meningitidis extracts had activities corresponding to TMP and TDP kinases, but whole cells which were exposed to TMP‐methyl‐3H did not seem to take up significant amounts of this compound. The findings indicate that N. meningitidis lacks the enzyme systems that are known to mediate a specific incorporation of thymine or thymidine into DNA.

Effect of 2-aminopurine and 2-aminopurine 2′-deoxyriboside on nucleic acid synthesis in ehrlich ascites cells in vitro

2-Aminopurine 2′-deoxyriboside has been prepared by transfer of 2′-deoxyribose from 2′-deoxythymidine to 2-aminopurine catalysed by nucleoside deoxyribosyltransferase from L. helveticus. 2-Aminopurine and 2-aminopurine 2′-deoxyriboside (in concentration of 2 μmoles/ml) inhibited the incorporation of 32P i and adenine-8- 14C into DNA of Ehrlich ascites tumor cells in vitro. Incorporation of 32P i into RNA was slightly inhibited by the two analogues, but the incorporation of adenine-8- 14C was not. 2-Aminopurine and 2-aminopurine 2′-deoxyriboside was recovered quantitatively from the acid soluble fraction of Ehrlich cells incubated with the compounds. No ribotides or deoxyribotides could be detected by the paper-chromatographic method used. 2-Aminopurine is not a substrate for a purine nucleoside phosphorylase from calf spleen. 5-Phosphoribosyl-2-aminopurine was formed in low yield from 2-aminopurine and 5-phosphoribosyl-1-pyrophosphate when incubated with a cell-free extract of Ehrlich cells. 2-Aminopurine 2′-deoxyriboside is not deaminated by an adenosine deaminase isolated from calf intestine, but is the strongest competitive inhibitor hitherto found of this enzyme.

Deoxyribosyl-8-azaadenine. Its deamination to deoxyribosyl-8-azahypoxanthine and effect on nucleic acid synthesis in Ehrlich ascites cells in vitro

Deoxyribosyl-8-azaadenine has been prepared by deoxyribose transfer from thymidine or deoxyinosine to 8-azaadenine with nucleoside deoxyribosyltransferase (EC 2.4.2.6) from Lactobacillus helveticus. Deoxyribosyl-8-azahypoxanthine was prepared by enzymatic deamination of deoxyribosyl-8-azaadenine. Deoxyribosyl-8-azaadenine is substrate for adenosine deaminase (EC 3.5.4.4) with a K m value of 125·10 −6 and a v max about 4 times that of deoxyadenosine. Deoxyribosyl-8-azaadenien and deoxyribosyl-8-azahypoxanthine have no effect on deoxyribonucleic acid and ribonucleic acid synthesis in Ehrlich ascites cells in vitro. Deoxyribosyl-8-azaadenine is rapidly deaminated in the tumor cells, but 8-azaadenine-deoxyribotides could not be detected. Deoxyribosyl-8-azahypoxanthine is neither cleaved in Ehrlich cells nor by purine nucleoside phosphorylase (EC 2.4.2.1).

Deoxyribosyl Transfer: I. THYMIDINE PHOSPHORYLASE AND NUCLEOSIDE DEOXYRIBOSYLTRANSFERASE IN NORMAL AND MALIGNANT TISSUES

Deoxyribosyl Transfer: I. THYMIDINE PHOSPHORYLASE AND NUCLEOSIDE DEOXYRIBOSYLTRANSFERASE IN NORMAL AND MALIGNANT TISSUES