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

Nucleoside phosphorylases (NPases) were found to be induced in Enterobacter aerogenes DGO-04, and cytidine and cytidine 5′-monophosphate (CMP) were the best inducers. Five mmol/L to fifteen mmol/L cytidine or CMP could distinctly increase the activities of purine nucleoside phosphorylase (PNPase), uridine phosphorylase (UPase) and thymidine phosphorylase (TPase) when they were added into medium from 0 to 8 h. In the process of enzymatic synthesis of adenine arabinoside from adenine and uracil arabinoside with wet cells of Enterobacter aerogenes DGO-04 induced by cytidine or CMP, the reaction time could be shortened from 36 to 6 h. After enzymatic reaction the activity of NPase in the cells induced remained higher than that in the cells uninduced.

Structural Insight into the Binding of Hypoxanthine with Purine Nucleoside Phosphorylase from Streptococcus mutants

Purine nucleoside phosphorylase is a key enzyme in the purine-salvage pathway and an attractive target for drug design. The crystal structure of Streptococcus mutants purine nucleoside phosphorylase（Smu PNP） has been solved by molecular replacement at 1.80 resolution and refined to R factors of 19.9%/23.7%（Rcryst/Rfree） . Sequence alignment and structural comparison show that Smu PNP has more similarity with PNPs isolated from human and malarial sources than the bacterial PNPs. The structure complexed with hypoxanthine（HPA） and sulfate ion was solved at 2.24A resolution and refined to R factors of 21.6%/24.1%（Rcryst/Rfree） . It is interesting to note that the resulting electron density indicated the product,HPA,presents in the active site although inosine was included in the crystallization mixture with Smu PNP. Asn233 and Glu191 are the important residues for ligand binding and recognition. Comparison with PNPs from different species gives detailed information about binding of small molecules on the active site,which is important for the studies of enzymatic mechanism and rational design of specific inhibitors for PNPs.

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

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-l-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℃. In addition, several other nucleosides and nucleoside derivatives were efficiently synthesized using bacterial strains co-expressing these recombinant enzymes.

Two-step Enzymatic Synthesis of Guanine Arabinoside

The chemical synthesis of Guanine arabinoside （ara-G） is extremely complex, time-consuming, and seriously polluted. A two-step enzymatic synthesis process was developed to acquire ara-G easily. 2,6-Diaminopurine arabinoside （ara-DA） was first synthesized with purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase produced by Enterobacter aerogenes DGW-07. The conversion yield of ara-DA could reach above 90% when the reaction liquid contained 30 mmol·L^-1 uracil arabinoside as arabinose donor, 10 mmol·L^- 1 2,6-diaminopurine as arabinose acceptor in pH 7.0 20 mmol·L^-1 phosphate buffer, and reacted at 60℃ for 48h. Then, ara-DA was effectively transformed into ara-G with adenylate deaminase produced by Aspergillus oryzae DAW-01. The total process had no complex separation and purification.