“…Interestingly, B. psychrosaccharolyticus whole cells have been also reported to display nucleoside 2 -deoxyribosyltransferase activity [24] and the analysis of its genome permitted the identification of the ndt gene that codifies a putative NDT [9]. Subsequently, this gene was cloned and overexpressed and the recombinant protein produced and immobilized [25,39].…”
Structure-function relationships of a novel 2 -deoxyribosyltransferase from the psychrotolerant bacterium Bacillus psychrosaccharolyticus (BpNDT) have been exhaustively studied by biochemical and high resolution crystallographic analyses. Despite BpNDT exhibiting some structural features characteristic of cold-adapted enzymes such as localized flexibility in critical loops, its biochemical properties are typical of mesophilic enzymes. BpNDT is a highly symmetrical homohexamer with tightly associated subunits that possesses flexible and short loops bordering the active sites. The catalytic center is essentially identical to that of other mesophilic homologues. Moreover, BpNDT shows that it is a mesophilic-like enzyme since it is not heat-labile and exhibits an apparent unfolding temperature (T m ) of 49 • C, being active during 96 h at 40 and 50 • C. Finally, BpNDT synthesizes natural and modified nucleosides, with preference for purines as acceptors and pyrimidine nucleosides as donors. Remarkably, the synthesis of several therapeutic nucleosides has been efficiently carried out. In this sense, 5-hydroxymethyl-2 -deoxyuridine (5-HMdUrd), 7-deaza-6-hydroxypurine-2 -deoxyriboside (7-DHPdRib) and theophylline-2 -deoxyriboside were synthesized for the first time by an NDT enzyme, showing the biotechnological interest of BpNDT.
“…Interestingly, B. psychrosaccharolyticus whole cells have been also reported to display nucleoside 2 -deoxyribosyltransferase activity [24] and the analysis of its genome permitted the identification of the ndt gene that codifies a putative NDT [9]. Subsequently, this gene was cloned and overexpressed and the recombinant protein produced and immobilized [25,39].…”
Structure-function relationships of a novel 2 -deoxyribosyltransferase from the psychrotolerant bacterium Bacillus psychrosaccharolyticus (BpNDT) have been exhaustively studied by biochemical and high resolution crystallographic analyses. Despite BpNDT exhibiting some structural features characteristic of cold-adapted enzymes such as localized flexibility in critical loops, its biochemical properties are typical of mesophilic enzymes. BpNDT is a highly symmetrical homohexamer with tightly associated subunits that possesses flexible and short loops bordering the active sites. The catalytic center is essentially identical to that of other mesophilic homologues. Moreover, BpNDT shows that it is a mesophilic-like enzyme since it is not heat-labile and exhibits an apparent unfolding temperature (T m ) of 49 • C, being active during 96 h at 40 and 50 • C. Finally, BpNDT synthesizes natural and modified nucleosides, with preference for purines as acceptors and pyrimidine nucleosides as donors. Remarkably, the synthesis of several therapeutic nucleosides has been efficiently carried out. In this sense, 5-hydroxymethyl-2 -deoxyuridine (5-HMdUrd), 7-deaza-6-hydroxypurine-2 -deoxyriboside (7-DHPdRib) and theophylline-2 -deoxyriboside were synthesized for the first time by an NDT enzyme, showing the biotechnological interest of BpNDT.
“…On the contrary, the salvage pathway is composed by a group of reutilization routes by which the cell can satisfy its purine requirements from endogenous and/or exogenous sources of preformed purines. In this regard, numerous enzymes from purine salvage pathway have become valuable catalysts for mono or multi-enzymatic synthesis of nucleosides and nucleotides, such as nucleoside kinases (NKs) [12][13][14][15], phosphoribosyltransferases [7,[9][10][11], nucleoside phosphorylases [4,8,16,17], 2′-deoxyribosyltransferases [5,[18][19][20], among others.…”
Section: Introductionmentioning
confidence: 99%
“…2.4.2.6) catalyze the transglycosylation reaction between a 2′-deoxynucleoside donor and a nucleobase acceptor. According to their substrate specificity, NDTs can be classified into two classes: type I (PDT), specific for purines (Pur↔Pur), and type II (NDT), which catalyze the transfer between purines and/or pyrimidines (Pur↔Pur, Pur↔Pyr, Pyr↔Pyr) [5,19,20] (Figure 1). Multi-enzymatic system LdNDT/TtHGXPRT.…”
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.
“…First of all, a derivative based on ionic adsorption of LaNDT (IDA-LaNDT) was obtained and it was able to yield 70% of DAC conversion in only 15 min, showing a high productivity. Thus a signi cant improvement in the productivity previously reported by other groups using this kind of enzyme was achieved (Fernández-Lucas et al 2011;Fresco-Taboada et al 2016). However, the industrial application of enzymes is feasible only if they are stabilized.…”
A novel IDA-LaNDT derivative was able to reach the highest productivity in the biosynthesis of a well-known antitumoral agent called decitabine. However, the combination of two simple and inexpensive techniques such as ionic absorption and gel entrapment with the incorporation of a bionanocomposite such as bentonite significantly improved the stability of this biocatalyst. These modifications allowed the enhancement of storage stability (for at least 18 months), reusability (400 h of successive batches without significant loss of its initial activity), and thermal and solvent stability with respect to the non-entrapped derivative. Moreover, reaction conditions were optimized by increasing the solubility of 5-aza by dilution with dimethylsulfoxide. Therefore, a scale-up of the bioprocess was assayed using the developed biocatalyst, obtaining 221 mg/L.h of DAC. Finally, green parameters were calculated using the nanostabilized biocatalyst, whose results indicated that it was able to biosynthesize DAC by a smooth, cheap, and environmentally friendly methodology.
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