Induction of nucleoside phosphorylase in Enterobacter aerogenes and enzymatic synthesis of adenine arabinoside

Wei, Xiaokun; Ding, Qingbao; Zhang, Lu; Yong-li, Guo; Ou, Lin; Wang, Changlu

doi:10.1631/jzus.b0710618

Cited by 9 publications

(4 citation statements)

References 19 publications

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“…Because of the broader substrate specificity, the latter group of enzymes is especially interesting for synthetic applications. Although many PNPs have been extensively studied , examples of ‘high molecular mass PNPs’ derived from thermophiles are limited to those from Geobacillus stearothermophilus (GsPNP, punB gene) and Enterobacter aerogenes (EaPNP) ; the MTAP from Aeropyrum pernix (ApMTAP) , Sulfolobus solfataricus (SsMTAP) and Pyrococcus furiosus (PfMTAP) , whereby only GsPNP, EaPNP and ApMTAP were considered for synthetic applications. Besides, ApMTAP has been recently applied in synthesis reactions , however, the information about the enzyme's characteristics is not available.…”

Section: Introductionmentioning

confidence: 99%

Recombinant purine nucleoside phosphorylases from thermophiles: preparation, properties and activity towards purine and pyrimidine nucleosides

et al. 2013

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Thermostable nucleoside phosphorylases are attractive biocatalysts for the synthesis of modified nucleosides. Hence we report on the recombinant expression of three 'high molecular mass' purine nucleoside phosphorylases (PNPs) derived from the thermophilic bacteria Deinococcus geothermalis, Geobacillus thermoglucosidasius and from the hyperthermophilic archaeon Aeropyrum pernix (5′-methythioadenosine phosphorylase; ApMTAP). Thermostability studies, kinetic analysis and substrate specificities are reported. The PNPs were stable at their optimal temperatures (DgPNP, 55°C; GtPNP, 70°C; ApMTAP, activity rising to 99°C). Substrate properties were investigated for natural purine nucleosides [adenosine, inosine and their C2′-deoxy counterparts (activity within 50-500 UÁmg ) as well as 2′-deoxy-2′-fluoroadenosine (9) and its C2′-arabino diastereomer (10, within 0.01-0.03 UÁmg À1 ). Our results reveal that the structure of the heterocyclic base (e.g. adenine or hypoxanthine) can play a critical role in the phosphorolysis reaction. The implications of this finding may be helpful for reaction mechanism studies or optimization of reaction conditions. Unexpectedly, the diastereomeric 2′-deoxyfluoro adenine riboand arabino-nucleosides displayed similar substrate properties. Moreover, cytidine and 2′-deoxycytidine were found to be moderate substrates of the prepared PNPs, with substrate activities in a range similar to those determined for 2′-deoxyfluoro adenine nucleosides 9 and 10. C2′-modified nucleosides are accepted as substrates by all recombinant enzymes studied, making these enzymes promising biocatalysts for the synthesis of modified nucleosides. Indeed, the prepared PNPs performed well in preliminary transglycosylation reactions resulting in the synthesis of 2′-deoxyfluoro adenine ribo-and arabino-nucleosides in moderate yield (24%).Abbreviations Ade, adenine; Ado, adenosine; Ap, Aeropyrum pernix; Cyd, cytidine; dAdo, 2′-deoxyadenosine; dAdo 2′F , 2′-deoxy-2′-fluoroadenosine; dAdo 2′F , 9-(2-deoxy-2-fluoro--D-arabinofuranosyl)adenine; dAdo 2′NH2 , 2′-amino-2′-deoxyadenosine; dCyd, 2′-deoxycytidine; Dg, Deinococcus geothermalis; dIno, 2′-deoxyinosine; dIno 2′NH2 , 2′-amino-2′-deoxyinoinosine; dUrd 2′F , 1-(2-deoxy-2-fluoro--D-arabinofuranosyl) uracil; dUrd 2′F , 2′-deoxy-2′

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Section: Introductionmentioning

confidence: 99%

Recombinant purine nucleoside phosphorylases from thermophiles: preparation, properties and activity towards purine and pyrimidine nucleosides

et al. 2013

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“…The temperature had a great influence on ara‐C catalysis, and the conversion rate reached 68.1% at 50°C. The solubility of ara‐A in distilled water is low, and a higher temperature facilitates the dissolution of substrates and improves the conversion of enzymes (Wei et al, 2008). However, a temperature higher than 60°C also deactivated enzymes and was detrimental to enzyme performance (Figure 1j).…”

Section: Resultsmentioning

confidence: 99%

“…More interestingly, the cell length of strain M-Δ377 (1.82 ± 0.16 μm) was 0.09 μm shorter than that of strain M (1.91 ± 0.20 μm), indicating that large-scale gene editing reduced the cell size (Figure 5g). The reactions catalyzed by PNP1 and UP are reversible (Wei et al, 2008), and theoretically, the transfer of bases cannot be complete. After genome editing, the obtained conversion rate of whole-cell catalysis (67.4 ± 2.5%) was very close to that of pure-enzyme catalysis (72.5 ± 4.3%).…”

Section: Gene Deletion and Bacterial Growth And Sizementioning

confidence: 99%

Whole‐cell biosynthesis of cytarabine by an unnecessary protein‐reduced Escherichia coli that coexpresses purine and uracil phosphorylase

Ping

Ruxian

Mengping

et al. 2022

Biotech & Bioengineering

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Currently, whole-cell catalysts face challenges due to the complexity of reaction systems, although they have a cost advantage over pure enzymes. In this study, cytarabine was synthesized by purified purine phosphorylase 1 (PNP1) and uracil phosphorylase (UP), and the conversion of cytarabine from adenine arabinoside reached 72.3 ± 4.3%. However, the synthesis was unsuccessful by whole-cell catalysis due to interference from unnecessary proteins (UNPs) in cells. Thus, we carried out a large-scale gene editing involving 377 genes in the genome of Escherichia coli to reduce the negative effect of UNPs on substrate conversion and cytarabine production. Finally, the PNP1 and UP activities of the obtained mutant were increased significantly compared with the parental strain, and more importantly, the conversion rate of cytarabine by whole-cell catalysis reached 67.4 ± 2.5%. The lack of 148 proteins and downregulation of 783 proteins caused by gene editing were equivalent to partial purification of the enzymes within cells, and thus, we provided inspiration to solve the problem caused by UNP interference, which is ubiquitous in the field of whole-cell catalysis.

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“…Previous studies have reported that nucleosides and nucleoside derivatives, such as 5-methyluridine (Ishii et al, 1989), 5-fluorouridine (Hori et al, 1992), 2′-deoxyadenosine (Yokozeki and Tsuji, 2000), and adenine arabinoside (Wei et al, 2008), can be successfully synthesized enzymatically using wild-type nucleoside phosphorylase (NPase). However, these reactions required a large concentration of bacterial cells as the source of the enzyme, thus limiting the application of this approach.…”

Section: Introductionmentioning

confidence: 99%

Enzymatic synthesis of nucleosides by nucleoside phosphorylase co-expressed in Escherichia coli

Ding

Wei

et al. 2010

J. Zhejiang Univ. Sci. B

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Abstract:Nucleoside phosphorylase is an important enzyme involved in the biosynthesis of nucleosides. In this study, purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase were co-expressed in Escherichia coli and the intact cells were used as a catalyst for the biosynthesis of nucleosides. For protein induction, lactose was used in place of isopropyl β-D-1-thiogalactopyranoside (IPTG). When the concentration of lactose was above 0.5 mmol/L, the ability to induce protein expression was similar to that of IPTG. We determined that the reaction conditions of four bacterial strains co-expressing these genes (TUD, TAD, DUD, and DAD) were similar for the biosyntheses of 2,6-diaminopurine nucleoside and 2,6-diaminopurine deoxynucleoside. When the substrate concentration was 30 mmol/L and 0.5% of the recombinant bacterial cell volume was used as the catalyst (pH 7.5), a greater than 90% conversion yield was reached after a 2-h incubation at 50 °C. In addition, several other nucleosides and nucleoside derivatives were efficiently synthesized using bacterial strains co-expressing these recombinant enzymes.

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Induction of nucleoside phosphorylase in Enterobacter aerogenes and enzymatic synthesis of adenine arabinoside

Cited by 9 publications

References 19 publications

Recombinant purine nucleoside phosphorylases from thermophiles: preparation, properties and activity towards purine and pyrimidine nucleosides

Recombinant purine nucleoside phosphorylases from thermophiles: preparation, properties and activity towards purine and pyrimidine nucleosides

Whole‐cell biosynthesis of cytarabine by an unnecessary protein‐reduced Escherichia coli that coexpresses purine and uracil phosphorylase

Enzymatic synthesis of nucleosides by nucleoside phosphorylase co-expressed in Escherichia coli

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