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

Mikhailopulo, Igor A.

doi:10.2174/138527207780059330

Cited by 94 publications

(84 citation statements)

References 95 publications

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“…These molecules have been synthesized by different chemical methods (27); however, the necessary tedious protection and deprotection steps often lead to low yields and increased costs. Indeed, chemical methods usually increase the difficulty of obtaining products with correct stereo-and regioselectivity, generating secondary products (13,34,56). As an alternative to the synthesis approach, enzymatic synthesis performed under very mild conditions in a stereoand regiospecific manner (42,55) is a valuable option.…”

Section: Discussionmentioning

confidence: 99%

“…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.…”

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confidence: 99%

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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: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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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“…NAs and NMPs have been traditionally synthesized by chemical methods through multistep processes requiring protection and de-protection steps for the labile groups, and isolation in almost every step due to the poor regio-or stereoselectivity of the reactions [4][5][6][7][8]. These drawbacks lead to a high price of these valuable compounds, limiting their application.…”

Section: Introductionmentioning

confidence: 99%

“…In this context, the enzymatic synthesis of NAs and NMPs shows many advantages, such as one-pot reactions under mild conditions, high stereo-, and regioselectivity, and an environmentally friendly technology [4][5][6][7][8][9][10][11].…”

Section: Introductionmentioning

confidence: 99%

One-Pot Multi-Enzymatic Production of Purine Derivatives with Application in Pharmaceutical and Food Industry

et al. 2018

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Abstract:Biocatalysis reproduce nature's synthetic strategies in order to synthesize different organic compounds. Natural metabolic pathways usually involve complex networks to support cellular growth and survival. In this regard, multi-enzymatic systems are valuable tools for the production of a wide variety of organic compounds. Methods: The production of different purine nucleosides and nucleoside-5 -monophosphates has been performed for first time, catalyzed by the sequential action of 2 -deoxyribosyltransferase from Lactobacillus delbrueckii (LdNDT) and hypoxanthine-guanine-xanthine phosphoribosyltransferase from Thermus themophilus HB8 (TtHGXPRT). Results: The biochemical characterization of LdNDT reveals that the enzyme is active and stable in a broad range of pH, temperature, and ionic strength. Substrate specificity studies showed a high promiscuity in the recognition of purine analogues. Finally, the enzymatic production of different purine derivatives was performed to evaluate the efficiency of multi-enzymatic system LdNDT/TtHGXPRT. Conclusions: The production of different therapeutic purine nucleosides was efficiently catalyzed by LdNDT/TtHGXPRT. In addition, the resulting by-products were converted to IMP and GMP. Taking all of these features, this bioprocess entails an efficient, sustainable, and economical alternative to chemical synthetic methods.

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“…The enzymatic methods have several advantages, such as a high regio-and stereoselectivity, mild reaction conditions, and limited use of contaminant reagents and organic solvents. NPs are one of the most widely spread tools for the synthesis of these compounds since they catalyze the specific combination of nucleobases and sugars (5,17,22).…”

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confidence: 99%

Thermus thermophilus Nucleoside Phosphorylases Active in the Synthesis of Nucleoside Analogues

Almendros

Berenguer

Sinisterra³

2012

Appl Environ Microbiol

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b Cells extracts from Thermus thermophilus HB27 express phosphorolytic activities on purines and pyrimidine nucleosides. Five putative encoding genes were cloned and expressed in Escherichia coli, and the corresponding recombinant proteins were purified and studied. Two of these showed phosphorolytic activities against purine nucleosides, and third one showed phosphorolytic activity against pyrimidine nucleosides in vitro, and the three were named TtPNPI, TtPNPII, and TtPyNP, respectively. The optimal temperature for the activity of the three enzymes was beyond the water boiling point and could not be measured accurately, whereas all of them exhibited a wide plateau of optimal pHs that ranged from 5.0 to 7.0. Analytical ultracentrifugation experiments revealed that TtPNPI was a homohexamer, TtPNPII was a monomer, and TtPyNP was a homodimer. Kinetic constants were determined for the phosphorolysis of the natural substrates of each enzyme. Reaction tests with nucleoside analogues revealed critical positions in the nucleoside for its recognition. Activities with synthetic nucleobase analogues, such as 5-iodouracil or 2,6-diaminopurine, and arabinosides were detected, supporting that these enzymes could be applied for the synthesis of new nucleoside analogs with pharmacological activities.

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

Cited by 94 publications

References 95 publications

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

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

One-Pot Multi-Enzymatic Production of Purine Derivatives with Application in Pharmaceutical and Food Industry

Thermus thermophilus Nucleoside Phosphorylases Active in the Synthesis of Nucleoside Analogues

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