Phosphorylation of Nucleosides by the Mutated Acid Phosphatase from Morganella morganii

A novel nucleoside phosphorylation process using the food additive pyrophosphate as the phosphate source was investigated. The Morganella morganii gene encoding a selective nucleoside pyrophosphate phosphotransferase was cloned. It was identical to the M. morganii PhoC acid phosphatase gene. Sequential in vitro random mutagenesis was performed on the gene by error-prone PCR to construct a mutant library. The mutant library was introduced into Escherichia coli, and the transformants were screened for the production of 5′-IMP. One mutated acid phosphatase with an increased phosphotransferase reaction yield was obtained. With E. coli overproducing the mutated acid phosphatase, 101 g of 5′-IMP per liter (192 mM) was synthesized from inosine in an 88% molar yield. This improvement was achieved with two mutations, Gly to Asp at position 92 and Ile to Thr at position 171. A decreased K_m value for inosine was responsible for the increased productivity.     

Studies on 5′-Nucleotidase-Lacking Mutants Derived from Bacillus subtilis

Two 5′-nucleotidase-lacking mutants, R–42 and A–1, were derived from an adenine-requiring mutant, B. subtilis 1145–2–83, which has productivity of both inosine and hypoxanthine. Strain A–1 accumulated 5′-IMP as well as inosine and hypoxanthine, and strain R–42 accumulated 5′-IMP and 5′-GMP as well as inosine and hypoxanthine in their culture fluids. These mutants responded to either adenine or adenosine, but did not to 5′-AMP. This fact suggests that adenine or adenosine may be incorporated into the cells, but 5′-AMP may neither be incorporated into the cells nor be degraded during culture. 5′-GMP was converted to 5′-IMP, and 5′-AMP was phosphrylated to ADP in the growing culture of strain A–1.     

Studies on 5′-Nucleotidase-Lacking Mutants Derived from Bacillus subtilis

For the purpose of effective accumulation of 5′-MMP, mutants, whose 5′-IMP-dephosphorylating activities were lower than that of strain A-1 of B. subtilis capable of accumulating a small amount of 5′-IMP as well as inosine and hypoxanthine, were derived from inosine-producing strain 1145-2-83 and strain A-1.

As a result, several mutants different from one another in the level of 5′-IMP-dephos- phorylating activity were isolated. Any of them did not acquire high ability to accumulate 5′-IMP. The more the mutants lost 5′-IMP-dephosphorylating activity, the less they accumulated extracellular inosine. The loss of nucleotide-dephosphorylating activity in the adenine-requiring mutants resulted in a remarkable increase in the amount of adenine required. The accumulation of 5′-IMP was not repressed by the addition of adenine at the concentration enough to repress accumulation of inosine.     

A novel process of inosine 5′-monophosphate production using overexpressed guanosine/inosine kinase

A novel process for producing inosine 5′-monophosphate (5′-IMP) has been demonstrated. The process consists of two sequential bioreactions; the first is a fermentation of inosine by a mutant of Corynebacterium ammoniagenes, and the second is a unique phosphorylating reaction of inosine by guanosine/inosine kinase (GIKase). GIKase was produced by an Escherichia coli recombinant strain, MC1000(pIK75), which overexpressed the enzyme up to 50% of the total cellular protein. The overproducing plasmid, pIK75, which was randomly screened out from deletion plasmids with various lengths of intermediate sequence between the E. coli trpL Shine-Dalgarno sequence, derived from the vector plasmid, and the start codon of the GIKase structural gene. In pIK75, the start ATG was placed 16 bp downstream of the trpL Shine-Dalgarno sequence under the control of the E. coli trp promoter. Fermentation of inosine and its phosphorylation were sequentially performed in a 5-l jar fermenter. At the end of inosine fermentation by C. ammoniagenes KY13761, culture broth of MC1000(pIK75) was mixed with that of KY13761 to start the phosphorylating reaction. Inosine in the reaction mixture was stoichiometrically phosphorylated, and 91 mM 5′-IMP accumulated in a 12-h reaction. This new biological process has advantages over traditional methods for producing 5′-IMP. Received: 7 April 1997 / Received last revision: 18 July 1997 / Accepted: 27 July 1997     

Recognition of Ribose Moiety by Adenosine (Phosphate) Deaminase from Squid Liver

An adenosine (phosphate) deaminase from the squid liver had much lower activity for 5′-deoxyadenosine than that for adenosine, 2′-, or 3′-deoxyadenosine. 3′-IMP and inosine as well as purine riboside and adenine competitively inhibited the deamination of adenosine 3′ phenylphosphonate by the enzyme, but 5′-AMP and 5′-IMP did not. The enzyme deaminated the 5′-hydroxyl terminal adenosine residue in dinucleotides and trinucleotide, but not the 3′-hydroxyl terminal one in dinucleotides. The 5′-hydroxyl group of the ribose moiety was necessary for the substrate binding and catalytic activity of the squid enzyme. These results indicated that the recognition of ribose moiety in the substrate by the squid enzyme might be intermediate between those by adenosine deaminase and adenosine (phosphate) deaminase from microorganisms.     

5′-Nucleotidase Activity in Improved Inosine-producing Mutants of Bacillus subtilis

An inosine- and guanosine-producing strain, AJ11100, of Bacillus subtilis could not grow in the minimum medium supplemented with 50 µg of sulfaguanidine per ml. When sulfaguanidine resistant mutants were derived from AJ11100, the sulfaguanidine resistance was frequently accompanied by xanthine requirement. All the xanthine auxotrophic mutants required a large amount of xanthine for cell growth and inosine accumulation. Revertants were then derived from one of the xanthine auxotrophic mutants, AJ11101, and improved inosine producers were obtained. The best mutant, AJ11102, accumulated 20.6 g of inosine per liter.

Furthermore, enzyme activities of inosine 5′-monophosphate (IMP) dehydrogenase, 5′-nucleotidase and phosphoribosyl pyrophosphate (PRPP) amidotransferase were assayed to investigate why AJ11102 accumulated an increased amount of inosine. The results showed that the increase of specific activity of 5′-nucleotidase contributed much to the increased accumulation of inosine.     

Molecular Structure of Xanthocidin

In order to establish industrial production of 5′-inosinic acid (5′-IMP), a permeability mutant, KY13171, of Brevibacterium ammoniagenes, which accumulated 7 to 8 grams of 5′-IMP per liter and 4 to 6 grams of hypoxanthine (Hx) per liter (calculated as 5′-IMP), was improved by a genetical procedure. Further improved mutants were selected stepwise through repeating mutational work. The finally selected mutant. KY13369, accumulated 20 to 27 grams of 5′-IMP per liter, but not Hx.

Increased productivity of 5′-IMP and decreased productivity of Hx were not caused by the changes in 5′-IMP degrading activity, because these activities were not significantly different among the mutants. These results appear to indicate that the increased accumulation of 5′-IMP may be caused by the improvement in membrane permeability for 5′-IMP. However, the changes in phospholipid and fatty acid compositions were not enough to explain the increased permeability.     

Expression of the guanosine-inosine kinase gene from Exiguobacterium acetylicum in Corynebacterium ammoniagenes and phosphorylation of inosine to produce inosine 5′-monophosphate

The guanosine-inosine kinase gene (gsk) isolated from Exiguobacterium acetylicum was expressed in an ATP-regenerating strain, Corynebacterium ammoniagenes. In order to induce its expression, three promoters (those for the Escherichia coli tac, Escherichia coli trp, and Corynebacterium glutamicum odhA gene) with the corresponding ribosome-binding sequences were examined. The E. coli trp promoter was most efficient with regard to inducing the expression of gsk in C. ammoniagenes. Further, the resulting strain, which has both inosine kinase activity and ATP-regenerating activity, was used to induce the phosphorylation of inosine to produce inosine 5′-monophosphate (5′-IMP), which is widely used as a flavor enhancer; approximately 80 g of 5′-IMP/l was produced with a molar conversion ratio of 80%.     

Specific, reversible inactivation of yeast beta-hydroxy-beta-methylglutraryl-CoA reductase by CoA.

A non-stoichiometric material [Na₄(5′-IMP)₂·15H₂O]_0.2[Na₂(Pt(5′-IMP)₂ (trimethylenediamine))·13.5H₂O]_0.8 has been prepared and investigated by single-crystal X-ray methods and ¹H and ¹³C nmr spectroscopy. The compound is isomorphous with the monosodium and disodium salts of 5′-IMP and two Pt(II)-5′-IMP compounds previously reported to be non-stoichiometric. However, the structural changes in the packing motif of the 5′-IMP molecules induced on Pt(II) coordination are uniform only if the 5′-IMP complex containing (NH₃)₂Pt(II) is stoichiometric. Preliminary studies on the latter complex, synthesized in our laboratories, demonstrate that the complex is indeed stoichiometric.     

Production of Nucleic Acid-Related Substances by Fermentative Processes

An adenine-requiring mutant (KY7208) of Brevibacterium ammoniagenes ATTC 6872 was found to accumulate an appreciable quantity of IMP and hypoxanthine in the culture liquid.

Crystalline IMP was isolated from culture broth of KY7208 by the use of ion-exchange columns. The preparation obtained was definitely identified as 5′-IMP, based on the results on paperchromatography, UV and IR absorption spectra, and analyses of its hydrolysates.

Growth responses of this mutant were demonstrated to adenine and adenosine, but not to 5′-AMP, 3′-AMP and 5′-AMP.

Over 5 mg of IMP per ml of broth were produced by the organism in natural medium consisting of glucose, yeast extract, urea, high concentrations of phosphate and magnesim salts, and others. The chemical changes showed that hypoxanthine first accumulated in the earlier stage of fermentation, and IMP synthesis then took place with the disappearance of hypoxanthine in the later stage of fermentation.     

A comparative study on phosphotransferase activity of acid phosphatases from Raoultella planticola and Enterobacter aerogenes on nucleosides,sugars, and related compounds

Natural and modified nucleoside-5′-monophosphates and their precursors are valuable compounds widely used in biochemical studies. Bacterial nonspecific acid phosphatases (NSAPs) are a group of enzymes involved in the hydrolysis of phosphoester bonds, and some of them exhibit phosphotransferase activity. NSAP containing Enterobacter aerogenes and Raoultella planticola whole cells were evaluated in the phosphorylation of a wide range of nucleosides and nucleoside precursors using pyrophosphate as phosphate donor. To increase the productivity of the process, we developed two genetically modified strains of Escherichia coli which overexpressed NSAPs of E. aerogenes and R. planticola. These new recombinant microorganisms (E. coli BL21 pET22b-phoEa and E. coli BL21 pET22b-phoRp) showed higher activity than the corresponding wild-type strains. Reductions in the reaction times from 21 h to 60 min, from 4 h to 15 min, and from 24 h to 40 min in cases of dihydroxyacetone, inosine, and fludarabine, respectively, were obtained.     

Cyclic AMP phosphodiesterase activity at the external surface of intact skeletal muscles and stimulation of the enzyme by insulin

Small intact frog skeletal muscles were exposed to radioactively labeled adenosine 3′,5′-cyclic monophosphate (cAMP) during incubation in frog Ringer's solution buffered with Tris (RT). The fate of the nucleotide was followed by measuring the products in the incubation media. Paper chromatography was used for the separation and identification of these products; the amounts were measured using liquid scintillation spectrometry. It was found that cAMP was degraded to AMP, which was then converted to IMP and, to some extent, inosine. The degradation of cAMP to AMP was markedly inhibited by theophylline (10 mM) suggesting the presence of cAMP phosphodiesterase activity at the muscle surface. Kinetic studies of enzyme activity in situ revealed two apparent K_m values: 0.33 μm and 55 μm. Insulin (0.3 unit/ml) increased the phosphodiesterase activity at concentrations of cAMP ranging from 2 to 17 μm. The possible roles of the surface phosphodiesterase were discussed.     

Differentiation between Several Types of Phosphohydrolases in Light Microsomes of Corn Roots

The phosphohydrolase activity of a light microsomal fraction isolated from corn roots (Zea mays L. cv LG 55) was investigated. The fraction, which appears to be enriched in endoplasmic reticulum and Golgi membranes, has ATPase and pyrophosphatase activities that hydrolyze ATP and pyrophosphate at an optimum pH of 7.0, with K_m values of about 160 and 240 micromolar and with V_max values of about 200 and 50 nanomoles substrate hydrolyzed per milligram protein per minute, respectively. These enzymes differ in their sensitivity to anions and inhibitors. The ATPase is stimulated by sulfate anions, whereas pyrophosphatase is inhibited by molybdate. Furthermore, the simultaneous addition of ATP and pyrophosphate to the reaction medium increases phosphohydrolysis, suggesting that separate enzymes are operating in the membranes. We also observed that pyrophosphate competitively inhibits the ATPase, whereas ATP has no significant effect on the pyrophosphatase. On the other hand, we observed a detergent-stimulated, molybdate-insensitive inosine diphosphatase activity which, in the native state, hydrolyzes inosine diphosphate with a K_m of about 700 micromolar and a V_max of about 450 nanomoles inosine diphosphate hydrolyzed per milligram protein per minute. In the solubilized form, the enzyme appears to be fully active, exhibiting lower K_m values to hydrolyze inosine diphosphate. Furthermore, we found that native inosine diphosphatase is inhibited either by ATP or pyrophosphate, whereas inosine diphosphate inhibits the ATPase, but has no significant effect on the pyrophosphatase. It appears that inosine diphosphate is a positive modulator of the inosine diphosphatase, whereas ATP and pyrophosphate act as negative modulators of this enzyme.     

Exploration of the 5-bromopyrimidin-4(3H)-ones as potent inhibitors of PDE5

The substituents both at the 6-position of the 5-bromopyrimidinone ring and at the 5′-position of the phenyl ring of 5-bromopyrimidin-4(3H)-ones were explored. 5-Bromo-6-isopropyl-2-(2-propoxy-phenyl)pyrimidin-4(3H)-one was identified as a new scaffold for potent PDE5 inhibitors. The crystal structures of PDE5/2e and PDE5/10a complexes provided a structural basis for the inhibition of 5-bromopyrimidinones to PDE5. In addition, it was also found that there is a great tolerance for the substitution at the 5′-position of the phenyl ring of 5-bormopyrimidinones and the resulted compound 13a has the highest inhibition activity to PDE5 (IC₅₀, 1.7 nM).     

Crystal and molecular structure of [Cu(5'-inosine monophosphate) (dipyridylamine) (H2O)]2.4H2O.

The ternary complex [Cu(5′-IMP)(dpa)(H₂O)]₂ has been prepared and its structure analyzed by x-ray diffraction. It has a dimeric structure in which the 5′-IMP ligands coordinate solely through their phosphate groups. This geometry is in marked contrast to that of another $\text{Cu}\text{5′-}\text{IMP}$ ternary complex, [Cu(5′-IMPH)(bipy)(H₂O)₂]⁺, which shows metal binding through the purine base rather than the phosphate group.     

Glycosylations of Inosine and Uridine Nucleoside Bases and Synthesis of the New 1-(β-D-Glucopyranosyl)-Inosine-5′, 6″-diphosphate

Abstract

Gluco- and ribosylation of the bases of sugar protected inosine and uridine were investigated, obtaining only adducts with β-configuration at the new glycosidic carbon; stereospecific insertion of a sugar moiety at the 1-N of inosine was achieved either using a Mitsunobu approach (for ribosylation) or by direct coupling of 1-δ-bromoglucose 13 with 2′,3′,5′-tri-O-acetylinosine for glucosylation. 1-(β-D-glucosyl)-inosine, chosen as starting substrate for glucosylated analogs of cyclic IDP-ribose, was phosphorylated at the primary hydroxyls and tested in intramolecular pyrophosphate bond formation.     

Bacterial Synthesis of Nucleotides

Inosine-5′, 2′(or 3′)-diphosphate was prepared by incubating 5′-IMP and p-nitrophenyl-phosphate with the bacteria characterized to phosphorylate at C_{3′ (&2′)}, or, on the contrary, by incubating 2′-IMP and a donor with the others capable of synthesizing 5′-nucleotide, via their phosphoryl transfer reactions.

Formation of the 5′, 2′(or 3′)-diphosphates of guanosine, cytidine, and uridine was also demonstrated to be carried out under the same relationship between nucleotide isomer as an acceptor and specificities of bacterial phosphotransferases, as observed in the phosphorylation of adenylic acid isomers, while 5′-dTMP was phosphorylated by both groups of bacteria.     

Efficient production of deoxynucleoside-5′-monophosphates using deoxynucleoside kinase coupled with a GTP-regeneration system

Deoxynucleoside-5′-monophosphates (5′-dNMPs) are the basic components of DNA and are widely used in medicine and as chemical and biochemical reagents. A large amount of effort has been expended to obtain 5′-dNMPs of high quality and at a low cost. However, these procedures are inefficient and inconvenient. In this study, deoxyadenosine-5′-monophosphate (5′-dAMP), 2,6-diaminopurine deoxynucleoside-5′-monophosphate (5′-dDAMP), and deoxycytidine-5′-monophosphate (5′-dCMP) were biosynthesized using recombinant N-deoxyribosyltransferase II (NDT-II), deoxycytidine kinase, and acetate kinase in a one-pot reaction system. The ndt-II gene from Lactobacillus delbrueckii, dck from Bacillus subtilus, and ack from Escherichia coli K12 were overexpressed in E. coli BL21 (DE3). Thymidine was used as the deoxyribose donor; GTP was used as the phosphate donor, and acetyl phosphate was used to regenerate GTP. Under optimized conditions, each 10 mM adenine, 10 mM 2,6-diaminopurine, or 10 mM cytosine were converted into 9.01 mM 5′-dAMP, 8.68 mM 5′-dDAMP, or 6.23 mM 5′-dCMP, respectively. The high yield indicated that this process of biosynthesis of 5′-dAMP, 5′-dDAMP, or 5′-dCMP was efficient and economical, and this one-pot system may also potentially be used for the preparation of other types of 5′-dNMPs.     

l-Serine production using a resting cell system of Hyphomicrobium strains

Twenty-six strains of methylotrophic hyphomicrobia were examined for their ability to produce l-serine from methanol and glycine in a resting cell reaction. l-Serine productivity of over 5 mg/ml was observed in 7 strains, and Hyphomicrobium sp. NCIB10099 was found to exhibit the highest productivity. Under optimized conditions using this bacterium, 45 mg/ml l-serine was produced from 100 mg/ml glycine and 88 mg/ml methanol in 3 d. The high l-serine degrading activity of the bacterium was entirely suppressed by adding an appropriate amount of CdCl₂ (ca. 1 mM), resulting in an enhanced conversion ratio of glycine to l-serine (ca. 100% molar conversion).     

Calcium and cyclic AMP as possible mediators of neurohormone action in the hindgut of the cockroach, Leucophaea maderae

Adenosine 3′,5′-monophosphate (cyclic AMP) (10⁻⁵ g/ml) often caused a gradual increase in spotaneous contractile activity of the hindgut of the cockroach, Leucophaea maderae, and on rare occasions it would evoke a hormone-like response. However, aminophylline (2·5 × 10⁻⁴ g/ml) was capable of mimicking the neurohormone, and a concentration of 2·5 × 10⁻⁵ g/ml potentiated the contractile response evoked by the neurohormone: these responses were blocked by either the presence of 1 mM manganous ion or in a high potassium solution (162 mM). Propranolol (10⁻⁶ g/ml) and dopamine (10⁻⁴ g/ml) suppressed both spontaneous contractile events and neurohormone action. Dopamine (5 × 10⁻⁶ g/ml) also blocked action potential generation as did propranolol at 10⁻⁴ g/ml.These results lead us to suppose that cyclic AMP might serve as a mediator of neurohormone action by increasing calcium transport across the surface membrane of muscle fibres. Caffeine (2·5 × 10⁻⁴ g/ml), like aminophylline, caused a hormone-like response in normal hindguts. Even when the visceral muscles of the hindgut were depolarized in 162 mM potassium solution (without calcium), caffeine was still capable of inducing a phasic response. However, the addition of 2 mM calcium to such potassium-depolarized preparations caused a gradual increase in muscle tonus and substantially potentiated the response to caffeine.Such findings clearly implicate calcium as the mediator of excitation-contraction coupling in visceral muscle. While the interactions between the neurohormone, cyclic AMP, and calcium seem to be largely associated with the surface membrane and action potential generation.