Nucleotide phosphorylases

Compound 25 (Fig. 18.9), a prodrug of 9-P-D-arabinofuranosyl guanine (26), was developed for the potential treatment of leukemia. Compound 24 is poorly soluble in water and its synthesis by conventional techniques is difficult. An enzymatic demethoxylation process was developed using adenosine deaminase (Mahmoudian et al., 1999, 2001). Compound 25 was enzymatically prepared from 6-methoxyguanine (27) and ara-uracil (28) using uridine phosphorylase and purine nucleotide phosphorylase. Each protein was cloned and overexpressed in independent Escherichia coli strains. Fermentation conditions were optimized for production of both enzymes and a co-immobilized enzyme preparation was used in the biotransformation process at 200 g/L substrate input. Enzyme was recovered at the end of the reaction by filtration and reused in several cycles. A more water soluble 5 -acetate ester of compound 26 was subsequently prepared by an enzymatic acylation process using immobilized Candida antarctica lipase in 1,4-dioxane (100 g/L substrate) with vinyl acetate as the acyl donor (Krenitsky et al., 1992). 

Radabaugh, T.R., Sampayo-Reyes, A., Zakharyan, R.A., Aposhian, H.V. (2002). Arsenate reductase II. Purine nucleotide phosphorylase in the presence of dihydrolipoic acid is a route for reduction of arsenate to arsenite in mammalian systems. Chem. Res. Toxicol. 15 692-8. 

Note PNP = Purine Nucleotide Phosphorylase HGPRT = Hypoxanthine-Guanine Phosphoribosyl Transferase... 

At very low values of EC, when AMP is elevated it is deaminated via AMP deaminase to inosine monophosphate (IMP). This further displaces the adenylate kinase reaction in the direction of ATP synthesis. The IMP is dephosphorylated by nucleotide phosphatase, and the inosine is phosphorylyzed via purine nucleotide phosphorylase, releasing hypoxanthine and ribose 1-phosphate. The latter is metabolized via the pentose phosphate pathway, and most of the carbon atoms enter glycolysis. Because this course of events depletes the overall adenine nucleotide pool, and hence the scope for ATP production in the longer term, it represents a metabolic last ditch stand by the cell to extract energy even from the energy currency itself ... 

In the purine degradation pathway IMP and GMP are dephosphorylated by nonspecified phosphatases to the corresponding nucleosides, which can leave the bacteria either spontaneously or facilitated by an exporter, for example, PbuE [290, 291]. Further degradation of the nucleosides to the nucleobases is catalyzed by the nucleotide phosphorylases PunA and DeoD [292]. The bases can be salvaged by Hpt-catalyzed reactions with PRPP to IMP and GMP. 

The base is directly ribosylated with PRPP by PT. The two enzymes concerned with purine salvage are adenine phosphoribosyl transferase (APRT), which catalyzes the formation of AMP, and hypoxanthine-guanine phosphoribosyl transferase (HGPRT), which catalyzes the formation of GMP as well as IMP, the precursor of GMP and AMP (Figure 6.58). Purine nucleotide phosphorylases (PNPases) can also... 

While they were in Busch s laboratory, Siebert and his associates [27] studied the intranuclear distribution of a number of enzymes known to be associated with the nuclear fraction (see below). Siebert concluded that RNA polymerase and ribonuclease were two nuclear enzymes primarily found in the nucleolus. Nucleotide phosphorylase and ATPase A were only in part associated with the nucleolus. Other nuclear enzymes were... 

Even before Kornberg demonstrated the mechanism of DNA synthesis, Ochoa and his group studied the steps involved in RNA synthesis. The requirements for that reaction in Ochoa s system were an enzyme (nucleotide phosphorylase), four diphosphonucleo-tides (ADP, GDP, CDP, UDP), magnesium, and a primer. The proper combination of primer, nucleotides, and enzyme permitted the synthesis of ribose polynucleotides of different base compositions, such as poly U, poly A, etc. 

All eukaryotic nascent mRNAs, except for some eukaryotic viral RNAs, contain 5 -end cap structures. The cap consists of an N-methylguanine nucleotide connected to the S end (nucleotide N) of the RNA by three phosphate groups, i.e., m G(5 )ppp(5 )N h The cap is added to the free 5 ends of mRNAs in the nucleus before the polymerase has transcribed more than 20 nt. The biogenesis of the 5 -end cap involves a series of enzymatic reactions catalyzed by nucleotide phosphorylase (pppN ppN -I- Pj), RNA guanylyltransferase (ppN -I- GTP GpppN -I- PPj), and RNA methyltransferase (GpppN -P AdoMet - m GpppN -P AdoHcy). 

---