Phosphohydrolases and the production of 5′-nucleotides by direct fermentation

A major problem involved in the direct fermentation of nucleotides is their breakdown by phosphohydrolases. Thus, adenine auxotrophs of most microorganisms produce hypoxanthine and/or inosine rather than inosine 5′-monophosphate (IMP) while guanine auxotrophs excrete xanthosine rather than xanthosine 5′-monophosphate (XMP). Examination of a Bacillus subtilis mutant producing hypoxanthine plus inosine revealed at least four phosphohydrolases, three of which could attack nucleotides. Even when the extracellular nucleotide phosphohydrolase was inhibited by Cu⁺² and its surface-bound alkaline phosphohydrolase was repressed and inhibited by inorganic phosphate, or removed by mutation, the breakdown products were still the only products of fermentation. Under these conditions, the third enzyme, a surface-bound non-repressible nucleotide phosphohydrolase was still active. It appears, at least in B. subtilis, that excretion is dependent upon breakdown by this enzyme and if hydrolysis does not occur, excretion of purine nucleotides is feedback inhibited by the resultant high intracellular IMP concentration. Corynebacterium glutamicum mutants, on the other hand, can excrete intact nucleotides, and direct fermentations for IMP, XMP, and GMP have been described. An examination of phosphohydrolases in a GMP-producing culture revealed no extracellular or surface enzymes. Disruption of the cells resulted in liberation of cellular phosphohydrolase activity with a substrate specificity remarkably similar to the flavorenhancing properties of the 5′-nucleotides. The order of decreasing susceptibility was GMP, IMP, XMP; AMP was not attacked.     

Production of guanosine-5'-monophosphate and inosine-5'-monophosphate by fermentation

A biotin-requiring coryneform bacterium which produces glutamic acid was mutated to adenine dependency. The adenine-requiring strain, which excreted insoine-5′-monophosphate (IMP), was further mutated to xanthine dependency. As expected, IMP was also excreted by this mutant. The mutant strain was reverted to xanthine independence in an attempt to obtain a culture with an altered IMP dehydrogenase which would be less sensitive to feedback inhibition by guanosine-5′-monophosphate (GMP). A revertant was obtained which produced GMP and IMP, each at 0.5 g per liter. The reversion to xanthine independence had resulted in a concomitant requirement for isoleucine, leucine, and valine. Further mutation to increased nutritional requirements led to culture MB-1802, which accumulated 1 g per liter each of GMP and IMP. Both nucleotides were isolated in pure form. The concentrations of GMP and IMP produced by MB-1802 were four times that of cytidylate, uridylate, or adenylate, indicating that the mechanism of GMP and IMP production was direct and not via ribonucleic acid breakdown.     

Production of Guanosine-5′-Monophosphate and Inosine-5′-Monophosphate by Fermentation

A biotin-requiring coryneform bacterium which produces glutamic acid was mutated to adenine dependency. The adenine-requiring strain, which excreted insoine-5′-monophosphate (IMP), was further mutated to xanthine dependency. As expected, IMP was also excreted by this mutant. The mutant strain was reverted to xanthine independence in an attempt to obtain a culture with an altered IMP dehydrogenase which would be less sensitive to feedback inhibition by guanosine-5′-monophosphate (GMP). A revertant was obtained which produced GMP and IMP, each at 0.5 g per liter. The reversion to xanthine independence had resulted in a concomitant requirement for isoleucine, leucine, and valine. Further mutation to increased nutritional requirements led to culture MB-1802, which accumulated 1 g per liter each of GMP and IMP. Both nucleotides were isolated in pure form. The concentrations of GMP and IMP produced by MB-1802 were four times that of cytidylate, uridylate, or adenylate, indicating that the mechanism of GMP and IMP production was direct and not via ribonucleic acid breakdown.     

The gua operon of Escherichia coli K-12: evidence for polarity from guaB to guaA

Guanine auxotrophs of Escherichia coli K-12 were isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methane sulfonate, or the acridine mustard ICR 372. guaA (xanthosine 5'-monophosphate [XMP] aminase-less) mutants were distinguished from guaB (inosine 5'-monophosphate [IMP] dehydrogenase-less) mutants by their growth response to xanthine and by enzyme assay. Mutations were classified as base substitutions or frameshift on the basis of mutagen-induced reversion patterns. All guaA strains, including three frameshift mutants, produced derepressed levels of IMP dehydrogenase when cultured with a growth-limiting concentration of guanine. The guaB strains were of two types: (i) those producing derepressed levels of XMP aminase, and (ii) those producing basal levels of XMP aminase when grown under conditions of guanine starvation. In the guaB strains of the second type, the expression of the adjacent guaA gene is reduced. It is proposed that this pleiotropic effect of some guaB mutations is a result of polarity. The orientation of polarity suggests the gene order "operator"-guaB-guaA. Gel diffusion studies with IMP dehydrogenase antiserum showed that strains carrying polar guaB mutations do not produce cross-reacting material (CRM). The remaining guaB mutants were either CRM(+) or CRM(-). Mapping the mutations by three-factor crosses showed that polar and nonpolar guaB sites are clustered in a small genetic region cotransducible with guaA. The relative positions of the guaB mutational sites established that the polar mutations lie within the structural gene for IMP dehydrogenase.     

Accumulation of 5′ guanine nucleotides by mutants of Brevibacterium ammoniagenes

5′Xanthylic acid was efficiently converted to 5′guanine nucleotides (5′GMP, 5′GDP, and 5′GTP) without being degraded to guanine via 5′GMP by decoyinine resistant mutants of strain KY 13315 which had been isolated from Brevibacterium ammoniagenes and was practically devoid of 5′nucleotide degrading activity. The concentration of phosphate in the medium showed a profound effect on the ratio of the accumulated 5′guanine nucleotides, making it possible to direct the fermentation towards 5′GMP or 5′GTP. A direct accumulation of 5′guanine nucleotides from carbohydrate was possible by mixed cultivation of a 5′XMP accumulating strain and a 5′XMP converting mutant. A maximum concentration of 9.67 mg of 5′guanine nucleotides per ml was obtained directly from glucose in such a mixed culture.     

Evidence for the involvement of cytosolic 5'-nucleotidase (cN-II) in the synthesis of guanine nucleotides from xanthosine

In this paper, we show that in vitro xanthosine does not enter any of the pathways known to salvage the other three main natural purine nucleosides: guanosine; inosine; and adenosine. In rat brain extracts and in intact LoVo cells, xanthosine is salvaged to XMP via the phosphotransferase activity of cytosolic 5'-nucleotidase. IMP is the preferred phosphate donor (IMP + xanthosine --> XMP + inosine). XMP is not further phosphorylated. However, in the presence of glutamine, it is readily converted to guanyl compounds. Thus, phosphorylation of xanthosine by cytosolic 5'-nucleotidase circumvents the activity of IMP dehydrogenase, a rate-limiting enzyme, catalyzing the NAD(+)-dependent conversion of IMP to XMP at the branch point of de novo nucleotide synthesis, thus leading to the generation of guanine nucleotides. Mycophenolic acid, an inhibitor of IMP dehydrogenase, inhibits the guanyl compound synthesis via the IMP dehydrogenase pathway but has no effect on the cytosolic 5'-nucleotidase pathway of guanine nucleotides synthesis. We propose that the latter pathway might contribute to the reversal of the in vitro antiproliferative effect exerted by IMP dehydrogenase inhibitors routinely seen with repletion of the guanine nucleotide pools.     

Isolation and characterization of guanine auxotrophs in Neurospora crassa

An ad-9 strain of Neurospora crassa was mutagenized with ethylmethane sulfonate (5%) and selected for guanine auxotrophy. The resultant double adenine plus guanine mutant was backcrossed with wild type and a single guanine auxotroph was isolated from the progeny. In vitro assays indicated that the mutant had GMP synthetase activity comparable with wild type, but was completely lacking of IMP dehydrogenase activity. The guanine requirement can therefore be explained by the mutant's inability to convert IMP to XMP. Another guanosine auxotroph was able to adapt and grow on minimal medium after 3 days. This mutant had GMP synthetase activity comparable with wild type but had only 10% of the IMP dehydrogenase activity of wild type, which may possibly explain its ability to grow on minimal medium after 3 days. It was confirmed that the two isolates are not allelic by crossing the two and recovering 25% wild-type progeny. Out isolate must therefore be designated gua-2.     

Production of Nucleic Acid-Related Substances by Fermentative Processes

The presence of psicofuranine in the fermentation medium caused the accumulation of a copious amount of 5′–XMP by Brevibacterium ammoniagenes. The accumulation of 5′–XMP in the medium was considered to be due to the inhibition of converting 5′–XMP to 5′–GMP by psicofuranine, which is known as a specific inhibitor of XMP aminase.

It was previously reported that in 5′–IMP fermentation with Br. ammoniagenes pantothenate and thiamine, in addition to biotin which was required for the growth of the microorganism, were exclusively required. This requirement for both vitamins was also observed in 5′–XMP production induced by the antibiotic.

The addition of manganese in excess to the fermentation medium promoted the bacterial growth greatly and inhibited IMP production, whereas XMP production induced by piscofuranine was not affected by the addition of excess manganese.

The accumulation of XMP induced by the antibiotic was completely suppressed by the presence of purine derivatives such as guanine, and xanthine derivatives, and partially by hypoxanthine.

5′–XMP was identified by chemical and enzymatic analyses and by UV absorption spectrum.     

Pentose Metabolism in Candida utilis

In attempts to obtain GMP producing strains, Brevibacterium ammoniagenes was treated with UV, N.T.G. or D.E.S. as a mutagen. Adenine-guanine requiring mutants were obtained from an adenine-requiring mutant of Brev. ammoniagenes, KY 3482–9 and two of them, presumably adenine-xanthine requiring mutants, were then reverted to mutants which required only adenine for their growth.

Although these revertants were not able to accumulate a copious amount of GMP, most of them and of adenine-guanine requiring mutants produced larger amounts of IMP than the parent adenine-requiring strain.

Effects of Mn²⁺ and purine bases in the medium on IMP production by these mutants were examined and IMP productivities of these mutants were compared with the parent strain under optimal conditions.

These mutagenic treatments were thus proved to be effective for the increase of de novo IMP production by Brev. ammoniagenes mutants.

Brevibacterium ammoniagenes ATCC 6872 accumulates 5′-GDP and -GTP, or 5′-ADP and -ATP together with GMP or AMP in nucleotide fermentation by salvage synthesis.

With cell free extract of this strain, transphosphorylating reactions of AMP or GMP were investigated.

ATP-AMP transphosphorylating enzyme(s) was partially purified to 21.7 fold with acid treatment, salting-out and column chromatography.

In ATP-AMP and ATP-GMP transphosphorylating reactins, optimal conditions were decided such as for concentrations of enzyme, of MgCl₂ and of phosphate donor, pH and cell age as the enzyme sources.

Specificities of phosphate donors and acceptors were examined with both the partially purified enzymes or the sonicate. AMP and GMP were phosphorylated by ATP rapidly, but IMP and XMP were not, therefore supporting our previous finding that Brev. ammoniagenes could not accumulated IDP, ITP, XDP and XTP in IMP and XMP fermentation, respectively.

Although ATP was the best donor for both AMP and GMP phosphorylations, other nucleoside triphosphates and PRPP were used as phosphate donors.

Furthermore, phosphorylation of ADP to ATP was investigated and possible mechanisms of nucleoside di- or triphosphates synthesis in the nucleotide fermentation were discussed.

From these results, it is suggested as a possible mechanism for nucleoside di- and triphosphate accumulation by Brev. Ammoniagenes, that a nucleoside monophosphate formed is phosphorylated to a nucleoside di-phosphate with ATP or other phosphate donors and then the nucleoside diphosphate is converted to a triphosphate with these phosphate donors.

Both AMP and GMP were transphosphorylated rapidly to the corresponding nucleoside-diphosphates and triphosphates by ATP and by other high energy phosphate compounds with cell free extracts of Brevibacterium ammoniagenes.

Some enzyme inhibitors, such as metals and PCMB were shown to inhibit the phosphorylations of AMP and GMP. Higher levels of ATP, ADP, GTP and GDP also inhibited the activity of the partially purified ATP-AMP transphosphorylating enzyme(s).

In guanine nucleotides fermentation by salvage synthesis with this strain, addition of these inhibitors to the medium increased the amounts of GMP and total guanine nucleotides accumulated.

On the contrary, supplement of xylene or of other organic solvents to the medium stimulated the accumulation of both GTP and total guanine compouuds in this fermentation. From enzymatic studies, these solvents are presumed to have the ability to change cell permeability.

Such findings give an effective method for controlling the amounts of nucleotides accumulated in these fermentations.     

Antitumor and Antiviral Properties of Penicillic Acid

The effects of adenine and (or) guanosine concentration on the accumulation of inosine, xanthosine, adenosine and succino-adenosine were studied with various purine auxotrophs of Bacillus subtilis K strain. Genetical derepression of the common pathway enzymes resulted in increase in the accumulation of inosine, xanthosine and adenosine. Co-operative repression system of a common pathway enzyme, succino-AMP lyase with respect to adenine and guanosine, was confirmed under the condition of the accumulation test. From these and the relating other studies it was concluded that the synthesis of AMP was regulated mainly by the inhibition of PRPP amidotransferase by AMP and secondly by the repression of the common pathway enzymes by adenine and guanosine, that the synthesis of GMP was regulated mainly by the inhibition and repression of IMP dehydrogenase by guanine derivatives and that GMP was synthesized in preference to AMP at the branch point, IMP.     

Isolation and characterisation of guanine auxotrophs in Saccharomyces cerevisiae.

Mutants of yeast which are auxotrophic for guanine have been isolated from two prototrophic haploid strains, one of which carried the suppressor of purine excretion, su-pur, and the other carried the alternative allele, su-pur+. The mutants were allocated to three genes, gual, gua2, and gua3, between which no close linkage was demonstrable. Mutants of all three genes were recessive and showed normal Mendelian segregation in crosses. The gene gual was shown by an in vivo enzyme assay procedure to specify guanosine 5'-phosphate (GMP) synthetase, the second enzyme involved in the biosynthesis of GMP from inosine 5'-phosphate (IMP). Mutants of this gene excrete large amounts of purine derivatives, predominantly xanthosine, into guanine-free, but not into guanine-supplemented, medium. The gene gau2 is probably involved in the biosynthesis of riboflavin from guanine nucleotides; the phenotype of these mutants suggests a possible interaction between aromatic amino acid metabolism and riboflavin biosynthesis. No role for gua3 can be assigned on the evidence so far available, but it is not involved in the specification of IMP dehydrogenase, the first enzyme involved in the synthesis of GMP and IMP.     

Crystal structure of Tritrichomonas foetus inosine monophosphate dehydrogenase in complex with the inhibitor ribavirin monophosphate reveals a catalysis-dependent ion-binding site

Inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step in GMP biosynthesis. The resulting intracellular pool of guanine nucleotides is of great importance to all cells for use in DNA and RNA synthesis, metabolism, and signal transduction. The enzyme binds IMP and the cofactor NAD(+) in random order, IMP is converted to XMP, NAD(+) is reduced to NADH, and finally, NADH and then XMP are released sequentially. XMP is subsequently converted into GMP by GMP synthetase. Drugs that decrease GMP synthesis by inhibiting IMPDH have been shown to have antiproliferative as well as antiviral activity. Several drugs are in use that target the substrate- or cofactor-binding site; however, due to differences between the mammalian and microbial isoforms, most drugs are far less effective against the microbial form of the enzyme than the mammalian form. The high resolution crystal structures of the protozoan parasite Tritrichomonas foetus IMPDH complexed with the inhibitor ribavirin monophosphate as well as monophosphate together with a second inhibitor, mycophenolic acid, are presented here. These structures reveal an active site cation identified previously only in the Chinese hamster IMPDH structure with covalently bound IMP. This cation was not found previously in apo IMPDH, IMPDH in complex with XMP, or covalently bound inhibitor, indicating that the cation-binding site may be catalysis-dependent. A comparison of T. foetus IMPDH with the Chinese hamster and Streptococcus pyogenes structures reveals differences in the active site loop architecture, which contributes to differences in cation binding during the catalytic sequence and the kinetic rates between bacterial, protozoan, and mammalian enzymes. Exploitation of these differences may lead to novel inhibitors, which favor the microbial form of the enzyme.     

Inosine-5′-Monophosphate Dehydrogenase Is a Rate-determining Factor for p53-dependent Growth Regulation

We have proposed that reduced activity of inosine-5′-monophosphate dehydrogenase (IMPD; IMP:NAD oxidoreductase, EC 1.2.1.14), the rate-limiting enzyme for guanine nucleotide biosynthesis, in response to wild-type p53 expression, is essential for p53-dependent growth suppression. A gene transfer strategy was used to demonstrate that under physiological conditions constitutive IMPD expression prevents p53-dependent growth suppression. In these studies, expression of bax and waf1, genes implicated in p53-dependent growth suppression in response to DNA damage, remains elevated in response to p53. These findings indicate that under physiological conditions IMPD is a rate-determining factor for p53-dependent growth regulation. In addition, they suggest that the impd gene may be epistatic to bax and waf1 in growth suppression. Because of the role of IMPD in the production and balance of GTP and ATP, essential nucleotides for signal transduction, these results suggest that p53 controls cell division signals by regulating purine ribonucleotide metabolism.     

The inhibition of nucleic acid synthesis by mycophenolic acid

1. Mycophenolic acid, an antibiotic of some antiquity that more recently has been found to have marked activity against a range of tumours in mice and rats, strongly inhibits DNA synthesis in the L strain of fibroblasts in vitro. 2. The extent of the inhibition of DNA synthesis is markedly increased by preincubation of the cells with mycophenolic acid before the addition of [(14)C]thymidine. 3. The inhibition of DNA synthesis by mycophenolic acid in L cells in vitro is reversed by guanine in a non-competitive manner, but not by hypoxanthine, xanthine or adenine. 4. The reversal of inhibition by guanine can be suppressed by hypoxanthine, 6-mercaptopurine and adenine. 5. Mycophenolic acid does not inhibit the incorporation of [(14)C]thymidine into DNA in suspensions of Landschütz and Yoshida ascites cells in vitro. 6. Mycophenolic acid inhibits the conversion of [(14)C]hypoxanthine into cold-acid-soluble and -insoluble guanine nucleotides in Landschütz and Yoshida ascites cells and also in L cells in vitro. There is some increase in the radioactivity of the adenine fraction in the presence of the antibiotic. 7. Mycophenolic acid inhibits the conversion of [(14)C]hypoxanthine into xanthine and guanine fractions in a cell-free system from Landschütz cells capable of converting hypoxanthine into IMP, XMP and GMP. 8. Preparations of IMP dehydrogenase from Landschütz ascites cells, calf thymus and LS cells are strongly inhibited by mycophenolic acid. The inhibition showed mixed type kinetics with K(i) values of between 3.03x10(-8) and 4.5x10(-8)m. 9. Evidence was also obtained for a partial, possibly indirect, inhibition by mycophenolic acid of an early stage of biosynthesis of purine nucleotides as indicated by a decrease in the accumulation of formylglycine amide ribonucleotide induced by the antibiotic azaserine in suspensions of Landschütz and Yoshida ascites cells and L cells in vitro.     

Mycobacterium tuberculosis IMPDH in Complexes with Substrates,Products and Antitubercular Compounds

Tuberculosis (TB) remains a worldwide problem and the need for new drugs is increasingly more urgent with the emergence of multidrug- and extensively-drug resistant TB. Inosine 5’-monophosphate dehydrogenase 2 (IMPDH2) from Mycobacterium tuberculosis (Mtb) is an attractive drug target. The enzyme catalyzes the conversion of inosine 5’-monophosphate into xanthosine 5’-monophosphate with the concomitant reduction of NAD⁺ to NADH. This reaction controls flux into the guanine nucleotide pool. We report seventeen selective IMPDH inhibitors with antitubercular activity. The crystal structures of a deletion mutant of MtbIMPDH2 in the apo form and in complex with the product XMP and substrate NAD⁺ are determined. We also report the structures of complexes with IMP and three structurally distinct inhibitors, including two with antitubercular activity. These structures will greatly facilitate the development of MtbIMPDH2-targeted antibiotics.     

Production of Nucleic Acid-Related Substances by Fermentative Processes

Enzymatic studies with Brevibacterium ammoniagenes ATCC 6872 demonstrated that 5-phosphoribose pyrophosphokinase and purinenucleotide pyrophosphorylase were involved in the nucleotide synthesis from purine base by ATCC 6872 and that its actual accumulation from base seemed to take place extracellularly through the action of the salvage enzymes leaked out of cells. Mn²⁺ deficiency and the simultaneous presence of pantothenate and thiamine, essential for efficient nucleotide accumulation, caused the extracellular leakage of the two enzymes with the simultaneous excretion of R5P. In the direct IMP fermentation with the adenine auxotroph, it was verified that hypoxanthine first produced de novo was reconverted into IMP extracellularly by the salvage enzymes as speculated previously.

A guanine-requiring mutant of Brevibacterium ammoniagenes ATCC 6872 accumulated a large amonnt of 5′-xanthosine-monophosphate (abbreviated as XMP).

The quantity of XMP accumulated by the strain was affected significantly by guanine levels in the medium. The suppression of XMP accumulation by an excessive addition of guanine compounds was recovered by the supply of casamino acids in the medium.

An enzyme in the pathway of de novo XMP synthesis, IMP dehydrogenase (IMP: NAD oxidoreductase, EC 1.2.1.14), was repressed and inhibited by guanine compounds.

The facts that an exogenous xanthine was not converted to XMP by the growing cells and that the activity of XMP-pyrophosphorylase was very low or deficient suggest that XMP accumulation by the strain would be probably due to the direct excretion of the nucleotide from the cells.     

Quantitative Analysis of Purine Nucleotides Indicates That Purinosomes Increase de Novo Purine Biosynthesis

Enzymes in the de novo purine biosynthesis pathway are recruited to form a dynamic metabolic complex referred to as the purinosome. Previous studies have demonstrated that purinosome assembly responds to purine levels in culture medium. Purine-depleted medium or 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) treatment stimulates the purinosome assembly in HeLa cells. Here, several metabolomic technologies were applied to quantify the static cellular levels of purine nucleotides and measure the de novo biosynthesis rate of IMP, AMP, and GMP. Direct comparison of purinosome-rich cells (cultured in purine-depleted medium) and normal cells showed a 3-fold increase in IMP concentration in purinosome-rich cells and similar levels of AMP, GMP, and ratios of AMP/GMP and ATP/ADP for both. In addition, a higher level of IMP was also observed in HeLa cells treated with DMAT. Furthermore, increases in the de novo IMP/AMP/GMP biosynthetic flux rate under purine-depleted condition were observed. The synthetic enzymes, adenylosuccinate synthase (ADSS) and inosine monophosphate dehydrogenase (IMPDH), downstream of IMP were also shown to be part of the purinosome. Collectively, these results provide further evidence that purinosome assembly is directly related to activated de novo purine biosynthesis, consistent with the functionality of the purinosome.