Amido Phosphoribosyltransferase Research Articles

Metabolic engineering of Bacillus subtilis for high-level production of uridine from glucose.

As an intermediate in drug synthesis, uridine has practical applications in the pharmaceutical field. Bacillus subtilis is used as a host to boost uridine yield by manipulating its uridine biosynthesis pathway. In this study, we engineered a high-uridine-producing strain of B. subtilis by modifying its metabolic pathways invivo. Overexpression of the aspartate ammonia-lyase (ansB) gene increased the relative transcriptional level of ansB in B. subtilis TD320 by 13·18 times and improved uridine production to 15·13 g l-1 after 72-h fermentation. Overexpression of the putative 6-phosphogluconolactonase (ykgB) gene increased uridine production by the derivative strain TD325 to 15·43 g l-1 . Reducing the translation of the amido phosphoribosyl transferase (purF) gene and inducing expression of the subtilisin E (aprE) gene resulted in a 1·99-fold increase in uridine production after 24 h shaking. Finally, uridine production in the optimal strain B. subtilis TD335, which exhibited reduced urease expression, reached 17·9 g l-1 with a yield of 314 mg of uridine g-1 glucose. To our knowledge, this is the first study to obtain high-yield uridine-producing B. subtilis in a medium containing only three components (80 g l-1 glucose, 20 g l-1 yeast powder, and 20 g l-1 urea).

Genome-Wide Mapping of Cytosine Methylation Revealed Dynamic DNA Methylation Patterns Associated with Sporophyte Development of Saccharina japonica.

Cytosine methylation plays vital roles in regulating gene expression and plant development. However, the function of DNA methylation in the development of macroalgae remains unclear. Through the genome-wide bisulfite sequencing of cytosine methylation in holdfast, stipe and blade, we obtained the complete 5-mC methylation landscape of Saccharina japonica sporophyte. Our results revealed that the total DNA methylation level of sporophyte was less than 0.9%, and the content of CHH contexts was dominant. Moreover, the distribution of CHH methylation within the genes exhibited exon-enriched characteristics. Profiling of DNA methylation in three parts revealed the diverse methylation pattern of sporophyte development. These pivotal DMRs were involved in cell motility, cell cycle and cell wall/membrane biogenesis. In comparison with stipe and blade, hypermethylation of mannuronate C5-epimerase in holdfast decreased the transcript abundance, which affected the synthesis of alginate, the key component of cell walls. Additionally, 5-mC modification participated in the regulation of blade and holdfast development by the glutamate content respectively via glutamine synthetase and amidophosphoribosyl transferase, which may act as the epigenetic regulation signal. Overall, our study revealed the global methylation characteristics of the well-defined holdfast, stipe and blade, and provided evidence for epigenetic regulation of sporophyte development in brown macroalgae.

In silico analysis of the amido phosphoribosyltransferase inhibition by PY873, PY899 and a derivative of isophthalic acid

Selectively decreasing the availability of precursors for the de novo biosynthesis of purine nucleotides is a valid approach towards seeking a cure for leukaemia. Nucleotides and deoxynucleotides are required by living cells for syntheses of RNA, DNA, and cofactors such as NADP(+), FAD(+), coenzyme A and ATP. Nucleotides contain purine and pyrimidine bases, which can be synthesized through salvage pathway as well. Amido phosphoribosyltransferase (APRT), also known as glutamine phosphoribosylpyrophosphate amidotransferase (GPAT), is an enzyme that in humans is encoded by the PPAT (phosphoribosyl pyrophosphate amidotransferase) gene. APRT catalyzes the first committed step of the de novo pathway using its substrate, phosphoribosyl pyrophosphate (PRPP). As APRT is inhibited by many folate analogues, therefore, in this study we focused on the inhibitory effects of three folate analogues on APRT activity. This is extension of our previous wet lab work to analyze and dissect molecular interaction and inhibition mechanism using molecular modeling and docking tools in the current study. Comparative molecular docking studies were carried out for three diamino folate derivatives employing a model of the human enzyme that was built using the 3D structure of Bacillus subtilis APRT (PDB ID; 1GPH) as the template. Binding orientation of interactome indicates that all compounds having nominal cluster RMSD in same active site's deep narrow polar fissure. On the basis of comparative conformational analysis, electrostatic interaction, binding free energy and binding orientation of interactome, we support the possibility that these molecules could behave as APRT inhibitors and therefore may block purine de novo biosynthesis. Consequently, we suggest that PY899 is the most active biological compound that would be a more potent inhibitor for APRT inhibition than PY873 and DIA, which also confirms previous wet lab report.

Effect of purine limitation caused by an amidophosphoribosyl transferase (purF) mutation on polyphosphate kinase 1 (ppk1) gene expression

Polyphosphate kinase (ppk) synthesizes polyphosphate (polyPn+1) from polyPn and ATP and also catalyses its reversal reaction. In the process of identifying stimuli that affect E. coli ppk transcription, we show that purine limitation activates the E. coli ppk promoter by using a single-copy E. coli ppk promoter-lacZ (ppkPEc-lacZ) fusion and Ez-Tn5 random mutagenesis. The ppkPEc-lacZ expression was greatly induced (seven to eleven fold higher depending on the media) in purF mutant cells (e.g. cvpA::Ez-Tn5 and ΔpurF mutants). This behavior seems to result from the purine starvation because the ppkPEc-lacZ expression in the purF mutant cells was recovered to the level of wild-type cells by the addition of any type of purines (adenine, guanine, adenosine, and guanosine). The ppkPEc-lacZ expression was also increased in other pur mutants but not as much as in the purF mutant cells. Interestingly, the ppkPEc-lacZ expression was not changed in mutants with defective pyrimidine specific genes (e.g. ΔpyrE and ΔthiC). Transcription of four other bacterial ppk promoters also increased in the purF mutant cells. This data imply that purine deficiency seems to be a common and good inducer in bacterial ppk gene expression. Here, we present a novel report about the effect of purine biosynthesis on ppk gene expression in bacteria.

Accumulation of 5-phosphoribosyl-1-pyrophosphate in human CCRF-CEM leukaemia cells treated with antifolates

Amido phosphoribosyltransferase (APRT) catalyzes the first step of the de novo biosynthesis of purine nucleotides, the conversion of 5-phosphoribosyl-1-pyrophosphate (PRPP) into 5-phosphoribosylamine (PRA). APRT is a valid target for development of inhibitors as anticancer drugs. We have developed a thin layer chromatographic assay for PRPP extracted from cells. Using coupling enzymes, PRPP with excess [2- 14 C ]orotate (OA) is quantitatively converted to [2- 14 C ]OMP and then [2- 14 C ]UMP with hydrolysis of the PPi. The reaction products are isolated on poly(ethyleneimine)-cellulose (PEI-C) chromatograms. Human CCRF-CEM leukaemia cells growing in culture have been exposed to a number of antifolates and their effects upon cellular levels of PRPP determined. The steady-state level of PRPP measured in CCRF-CEM cells was 102±11 μM. Following addition of an antifolate to a culture, accumulation of PRPP in cells indicates the degree of inhibition of APRT. In human CCRF-CEM leukaemia cells, lometrexol (LTX), 2,4-diamino-6-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydro-quinazoline (PY899), methotrexate (MTX), N α(4-amino-4-deoxypteroyl)- N δ-hemiphthaloyl- l-ornithine (PT523), piritrexim (PTX), metoprine, 2,4-diamino-6-(3,4,5-trimethoxyanilino)-methylpyrido[3,2- d]pyrimidine (PY873) and multitargeted antifolate, N-[4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3- d]pyrimidin-5-yl)ethyl]benzoyl]- l-glutamic acid (MTA) directly or indirectly induce inhibition of APRT indicated by time-courses for accumulation of PRPP to maximum values of 3–12-fold. These data indicate that LTX induces the most potent inhibition of APRT.

Purine and pyrimidine nucleotide synthesis and metabolism.

Purine and pyrimidine nucleotides are major energy carriers, subunits of nucleic acids and precursors for the synthesis of nucleotide cofactors such as NAD and SAM. Despite the obvious importance of these molecules, we still have much to learn about how these nucleotides are synthesized and metabolized by plants. Moreover, of the research that has been done in this area relatively little has used genetic analysis to evaluate the function(s) of specific enzymes. The pathways for the synthesis of nucleotides in plant cells are similar to those found in animals and microorganisms. This conclusion is based primarily on the results of studies using in vivo radiotracers, specific inhibitors of nucleotide synthesis and on analyses of the kinetic parameters of purified enzymes involved in nucleotide synthesis that are unlikely to have similar demands for purine and pyrimidine nucleotides have been used in this research. A more comprehensive understanding of the role(s) of specific nucleotide biosynthetic enzymes throughout plant development and factors that regulate their activity/expression is still lacking. Ultimately this information will explain how the requirements of different plants are met, such as those of ureide-producing legumes (Schubert and Boland, 1990) or those synthesizing caffeine (Suzuki et al., 1992; Ashihara and Crozier, 1999). There are two principal routes for the synthesis of nucleotides: the de novo and the salvage pathways (Figures 1 and ​and2,2, Figures 3 and ​and4,4, respectively). Using 5-phosphoribosyl-1-pyrophosphate (PRPP), the de novo pathway enzymes build purine and pyrimidine nucleotides from “scratch” using simple molecules such as CO2, amino acids and tetrahydrofolate. This route of nucleotide synthesis has a high requirement for energy as compared that of the salvage pathway. For example, five of the 12 steps of de novo purine synthesis require hydrolysis of ATP or GTP but only one salvage cycle reaction uses ATP. The enzymes of both of these biosynthetic routes are classified as “housekeeping” enzymes because they perform basic, cellular activities and are assumed to be present in low, constitutive levels in all cells. Whereas the de novo pathway is thought to reside in plastids, salvage cycle enzymes may be localized in more than one compartment. Open in a separate window Figure 1. De novo biosynthetic pathway of purine nucleotides in plants. Enzymes shown are: amido phosphoribosyltransferase, (2) GAR synthetase, (3) GAR formyl transferase, (4) FGAM synthetase, (5) AIR synthetase, (6) AIR carboxylase, (7) SAICAR synthetase, (8) adenylosuccinate lyase, (9) AICAR formyl transferase, (10) IMP cyclohydrolase, (11) SAMP synthetase, (12) adenylosuccinase, (13) IMP dehydrogenase, (14) GMP synthetase.

Growth factor-regulated expression of enzymes involved in nucleotide biosynthesis: a novel mechanism of growth factor action.

Keratinocyte growth factor (KGF) is a potent and specific mitogen for epithelial cells, including the keratinocytes of the skin. We investigated the mechanisms of action of KGF by searching for genes which are regulated by this growth factor in cultured human keratinocytes. Using the differential display RT-PCR technology we identified the gene encoding adenylosuccinate lyase [EC 4.3.2.2] as a novel KGF-regulated gene. Adenylosuccinate lyase plays an important role in purine de novo synthesis. To gain further insight into the potential role of nucleotide biosynthesis in the mitogenic effect of KGF, we cloned cDNA fragments of the key regulatory enzymes involved in purine and pyrimidine metabolism (adenylosuccinate synthetase [EC 6.3.4.4], phosphoribosyl pyrophosphate synthetase [EC 2.7.6.1], amidophosphoribosyl transferase [EC 2.4.2.14], hypoxanthine guanine phosphoribosyl transferase [EC 2.4.2.8] and the multifunctional protein CAD which includes the enzymatic activities of carbamoyl-phosphate synthetase II [EC 6.3.5.59], aspartate transcarbamylase [EC 2.1.3.2] and dihydroorotase [EC 3.5.2.3]). Expression of all of these enzymes was upregulated after treatment with KGF and also with epidermal growth factor (EGF), indicating that these mitogens stimulate nucleotide production by induction of these enzymes. To determine a possible in vivo correlation between the expression of KGF, EGF and the enzymes mentioned above, we analysed the expression of the enzymes during cutaneous wound repair, where high levels of these mitogens are present. Indeed, we found a strong mRNA expression of all of these enzymes in the EGF- and KGF-responsive keratinocytes of the hyperproliferative epithelium at the wound edge, indicating that their expression might also be regulated by growth factors during wound healing.

Metabolic effects of thiopurine derivatives against human CCRF-CEM leukaemia cells

Background and aims. To compare the metabolic effects induced by the anticancer drugs, 6-mercaptopurine (6-MP), 6-thioguanine (6-TG) and 6-methylmercaptopurine riboside (MMPR), which may inhibit the de novo biosynthesis of purine nucleotides or be mis-incorporated into DNA or RNA. Methods. Leukaemia cells were grown in culture, exposed to a thiopurine and cell extracts were analyzed for NTPs, dNTPs, drug metabolites and P-Rib-PP. Results. In leukaemia cells, 6-MP was converted to MPR-MP, thio-XMP, thio-GMP, thio-GDP and thio-GTP. Metabolites of 6-TG included thio-XMP, thio-GMP, thio-GDP and thio-GTP, while MMPR-MP was the only major metabolite of MMPR. MMPR (25 μM, 4 h) induced a 16-fold increase in P-Rib-PP and 6-MP (25 μM, 4 h) induced a delayed 5.2-fold increase. MPR-MP, thio-GMP and MMPR-MP are inhibitors of amido phosphoribosyltransferase from leukaemia cells with K i values of 114±7.10 μM, 6.20±2.10 μM and 3.09±0.30 μM, respectively. Conclusion. The nucleoside-5′-monophosphate derivatives of the 3 thiopurines inhibit amido phosphoribosyltransferase in growing leukaemia cells but there is also an initial inhibition of the further conversion of IMP in the pathway. In growing cells, MMPR acts solely as an inhibitor of de novo purine biosynthesis while 6-TG and to a lesser extent, 6-MP, are converted to significant concentrations of di- and tri-phosphate derivatives which may have other mechanisms of cytotoxicity.

Mechanisms of inhibition of amido phosphoribosyltransferase from mouse L1210 leukemia cells.

Amido phosphoribosyltransferase (amido PRTase) catalyses the first step of the pathway for de novo biosynthesis of purine nucleotides. The enzyme is subject to inhibition by purine nucleoside 5'-monophosphates (AMP, IMP, and GMP), by dihydrofolate polyglutamates, and by the antifolate piritrexim [Sant, M. E., Lyons, S. D., Phillips, L., & Christopherson, R. I. (1992) J. Biol. Chem. 267, 11038-11045). Using a coupled radioassay, we have determined the substrate dissociation constants as 80.4 +/- 13.2 microM for 5-phosphoribosyl 1-pyrophosphate (P-Rib-PP) and 421 +/- 193 microM for L-glutamine with P-Rib-PP bound first with positive cooperativity for interaction with a second site on the catalytically active dimer (interaction factor of 0.247 +/- 0.042). Analysis of inhibition patterns for amido PRTase shows that the antifolate piritrexim is a noncompetitive inhibitor bound with positive cooperativity at two allosteric sites of an inactive dimer with a dissociation constant of 66.0 +/- 17.8 microM for interaction with the free enzyme and an interaction factor of 0.187 +/- 0.113 with P-Rib-PP as the varied substrate. With L-glutamine as the varied substrate, a dissociation constant of 62.3 +/- 15.6 microM for interaction with the enzyme-P-Rib-PP complex and an interaction factor of 0.0958 +/- 0.0585 microM were obtained. AMP binds as a competitive inhibitor with respect to P-Rib-PP with a dissociation constant of 40.0 +/- 8.1 microM for interaction with the free enzyme and as a noncompetitive inhibitor with respect to L-glutamine with a dissociation constant of 16.4 +/- 5.2 mM for interaction with the enzyme-P-Rib-PP complex. Sucrose density gradient centrifugation of partially purified amido PRTase showed three molecular forms of the enzyme: an inactive tetramer (10.2 S) formed in the presence of AMP, an active dimer (6.7 S) formed with P-Rib-PP, and an inactive dimer (7.2 S) with piritrexim. The latter species may predominate in cells containing high levels of dihydrofolate polyglutamates.

Genetic evidence for 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) as a negative effector of cytochrome terminal oxidase cbb3 production in Rhizobium etli.

A Rhizobium etli Tn5mob-induced mutant (CFN035) exhibits an enhanced capacity to oxidize N,N,N',N', tetramethyl-p -phenylenediamine (TMPD), a presumptive indicator of elevated cytochrome c terminal oxidase activity. Sequencing of the mutated gene in CFN035 revealed that it codes for the amidophosphoribosyl transferase enzyme (PurF) that catalyzes the first step in the purine biosynthetic pathway. Two c-type cytochromes with molecular weights of 32 and 27 kDa were produced in strain CFN035, which also produced a novel CO-reactive cytochrome (absorbance trough at 553 nm), in contrast to strain CE3 which produced a single 32 kDa c-type protein and did not produce the 553 nm CO-reactive cytochrome. A wild-type R. etli strain that expresses the Bradyrhizobium japonicum fixNOQP genes, which code for the symbiotic cytochrome terminal oxidase cbb3, produced similar absorbance spectra (a trough at 553 nm in CO-difference spectra) and two c-type proteins similar in size to those of strain CFN035, suggesting that CFN035 also produces the cbb3 terminal oxidase. The expression of a R. etli fixN-lacZ gene fusion was measured in several R. etli mutants affected in different steps of the purine biosynthetic pathway. Our analysis showed that purF, purD, purQ, purL, purY, purK and purE mutants expressed three-fold higher levels of the fixNOQP operon than the wild-type strain. The derepressed expression of fixN was not observed in a purH mutant. The purH gene product catalyzes the conversion of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to 5-form-aminoimidazole-4-carboxamide ribonucleotide (FAICAR) and inosine. Supplementation with AICA riboside lowered the levels of fixN expression in the purF mutants. These data are consistent with the possibility that AICAR, or a closely related metabolite, is a negative effector of the production of the symbiotic terminal oxidase cbb3 in R. etli.

Inhibition of murine amido phosphoribosyltransferase by folate derivatives.

Amido phosphoribosyltransferase (PRTase) catalyses the first reaction of the pathway for de novo biosynthesis of purine nucleotides: Open image in new window

Purine metabolism in promyelocytic HL60 and dimethylsulphoxide-differentiated HL60 cells

Purine metabolism was studied in the human promyelocytic leukaemic cell line HL60 both before and after differentiation by dimethylsulphoxide (DMSO) treatment in vitro. DMSO-induced differentiation had no effect on the steady-state concentration of 5-phosphoribosylpyrophosphate (PRPP), even though the activity of the oxidative segment of the pentose phosphate pathway (OPP) increased two-fold and the activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were enhanced 3–6 times. [ 14C]-formate incorporation, on the other hand, showed that undifferentiated HL60 cells produced purines by the de novo pathway at six-fold the rate of the differentiated cells and at three times the rate measured in polymorphonuclear leukocytes (PMNs) separated from human blood. This is consistent with significantly higher activities (two to five-fold) of the key regulatory enzymes of the de novo purine synthesis pathway (PRPP synthetase, amidophosphoribosyl transferase and adenylosuccinate synthase) in the undifferentiated cells. Labelling the cells with [ 14C]-hypoxanthine showed that there was an active salvage pathway in the HL60 cells which remained unaltered by differentiation. The activities of the salvage enzymes were also the same, although hypoxanthine was taken up at twice the rate by the undifferentiated cells. Human PMNs had an active salvage pathway, but their rate was five times lower than that of the HL60 cells, consistent with much lower activities (three to eightfold) of the salvage pathway enzymes. These observations suggest that regulation of the synthesis and activities of the purine nucleotide synthesis enzymes may play an important role in the regulation of differentiation of myeloid cells.

Antifolates induce inhibition of amido phosphoribosyltransferase in leukemia cells.

The pathway for de novo biosynthesis of purine nucleotides contains two one-carbon transfer reactions catalyzed by glycinamide ribotide (GAR) and 5-aminoimidazole-4-carboxamide ribotide (AICAR) transformylases in which N10-formyltetrahydrofolate is the one-carbon donor. We have found that the antifolates methotrexate (MTX) and piritrexim (PTX) completely block the de novo purine pathway in mouse L1210 leukemia cells growing in culture but with only minor accumulations of GAR and AICAR to less than 5% of the polyphosphate derivatives of N-formylglycinamide ribotide (FGAR) which accumulate when the pathway is blocked completely by azaserine. This azaserine-induced accumulation of FGAR polyphosphates is completely abolished by MTX, indicating that inhibition of the pathway is at or before GAR transformylase (reaction 3; Lyons, S. D., and Christopherson, R. I. (1991) Biochem. Int. 24, 187-197). Three h after the addition of MTX (0.1 microM), cellular 5-phosphoribosyl-1-pyrophosphate has accumulated 3.4-fold while 6-methyl-mercaptopurine riboside (25 microM) induces a 6.3-fold accumulation. These data suggest that amido phosphoribosyltransferase catalyzing reaction 1 of the pathway is the primary site of inhibition. In support of this conclusion, we have found that dihydrofolate-Glu5, which accumulates in MTX-treated cells, is a noncompetitive inhibitor of amido phosphoribosyltransferase with a dissociation constant of 3.41 +/- 0.08 microM for interaction with the enzyme-glutamine complex in vitro. Folate-Glu5, MTX-Glu5, PTX, dihydrotriazine benzenesulfonyl fluoride, and AICAR also inhibit amido phosphoribosyltransferase.

Inhibition of Amido Phosphoribosyltransferase by Antifolates in Mouse L1210 Leukaemia Cells

Inhibition of Amido Phosphoribosyltransferase by Antifolates in Mouse L1210 Leukaemia Cells

Regulation of de novo purine biosynthesis in Chinese hamster cells.

Regulation of de novo purine biosynthesis was examined in two Chinese hamster cell lines, CHO and V79. De novo purine biosynthesis is inhibited at low concentrations of adenine. The mechanism of inhibition was studied using the RNA and protein synthesis inhibitors actinomycin D, cycloheximide, and azacytidine. Although all three inhibitors rapidly inhibited de novo purine biosynthesis in vivo, neither adenine nor the RNA and protein synthesis inhibitors could be found to have an effect in vitro on either phosphoribosylpyrophosphate (PRPP) synthetase or amido phosphoribosyltransferase, the first enzymes of the de novo pathway. However, in the presence of actinomycin D, cycloheximide, and azacytidine, there was a 50% or greater reduction in PRPP concentrations. This reduction in PRPP levels is correlated with a 2-fold increase in purine nucleotides in the acid-soluble pool. It is proposed that in the presence of the metabolic inhibitors there is an increase in nucleotide pools due to degradation of RNA, with a resulting feedback inhibition on de novo purine biosynthesis. In contrast to a previous report (Martin, D. W., Jr., and Owen, N. T. (1972) J. Biol. Chem. 247, 5477-5485), we could find no evidence for a repressor type mechanism in these cells.

Kinetics of amidophosphoribosyltransferase in intact tumor cells

A method for measuring amidophosphoribosyltransferase (5-phosphoribosylamine:pyrophosphate phosphoribosyltransferase, EC 2.4.2.14) activity in intact Ehrlich ascites tumor cells is described. It depends on the measurement of the initial rate of glutamine-dependent utilization of intracellular 5-phosphoribosyl-1-pyrophosphate ( P- ribose-PP ). This utilization of P- ribose-PP is due solely to amidotransferase activity, as it can be inhibited completely by 6-methylmercaptopurine ribonucleoside-5′-phosphate. Apparent Michaelis constants for amido phosphoribosyltransferase assayed in tumor cell extracts were 2 mM for glutamine and 1 mM for P- ribose-PP . In intact tumor cells, 2.5 mM intracellular P- ribose-PP gave half-maximal activity. Concentrations of glutamine in incubation media of greater than 1 mM gave maximal amidotransferase activity in intact cells. The apparent maximum amidotransferase activity was the same in intact tumor cells and cell extracts (approx. 0.30 μmole/min per ml packed cells).