Synthesis Of Inosine Research Articles

Improving the Phosphatase-Catalyzed Synthesis of 5′-Nucleotides: A Reaction Engineering Approach

5′-Phosphorylation of nucleosides is a reaction as important in nature and in industry as it is cumbersome to be performed. Whilst chemical phosphorylation relies on the use of harsh reagents, solvents, and conditions, as well as on the need for protection–deprotection steps, biocatalysis can be a tool to achieve one-step phosphorylation reactions, which are selective, protecting group-free, and occurring under mild and sustainable conditions. In this work, the wild-type non-specific acid phosphatase from Morganella morganii (PhoC-Mm) was expressed, purified, and used for the synthesis of inosine 5′-monophosphate (IMP), an important food additive, by using pyrophosphate (PPi) as an inexpensive phosphate donor in a fully aqueous medium at 30 °C. Via the fine-tuning of the reaction set-up taking into account the type of buffer, amount of PPi, mode/time of PPi addition, and enzyme and substrate concentration, PhoC-Mm could be used for catalyzing the phosphorylation of inosine (I) to IMP in a good yield and high purity (62% yield). The catalysis of the hydrolytic reaction direction, which is the primary function of phosphatases in nature, was here reversed to a certain extent by a reaction engineering approach, without the need for protein engineering strategies.

Transcriptome analysis in comparing carcass and meat quality traits of Jiaxing Black Pig and Duroc×Duroc×Berkshire×Jiaxing Black Pig crosses.

This study compares two typical strains: Chinese local excellent meat quality of Jiaxing Black (JXB) Pig and quadratic crossbred pig strain Duroc×Duroc×Berkshire×Jiaxing Black (DDBJ). It was found that between the two pig strains, carcass traits and meat quality traits differed significantly. This is exemplified by the leanness and dressing out percent of DDBJ that were significantly higher than JXB pigs of the same age (P<0.05) and the better growth rate of DDBJ pigs as to JXB pigs was shown by quantifying muscle proliferation and differentiation of longissimus dorsi muscle employing Hematoxylin and Eosin staining of longissimus dorsi muscle. Nutrients such as inosinic acid, intramuscular fat, and free amino acids in the longissimus dorsi muscle were significantly higher in JXB pigs than DDBJ pigs (p<0.0001); saturated fatty acids were higher in JXB than in DDBJ pigs (p=0.0097); essential amino acids and fresh taste amino acids (serine, glutamic acid, proline, glycine, alanine) of JXB pigs was higher than that of DDBJ pigs (p<0.0001) and amino acids in longissimus dorsi muscle of JXB pigs surpasses the amino acid concentration of DDBJ pigs (p<0.0001), thus showing the superiority of JXB in terms of meat quality. However, the content of polyunsaturated fatty acids, which is responsible for poor meat quality, was significantly higher in the longissimus dorsi muscle of DDBJ pig than JXB pigs (p<0.0001); RNA-seq analysis of 5 biological replicates from two of the strains was performed. The screening of 164 up-regulated genes and 183 down-regulated genes found in longissimus dorsi muscle of DDBJ was done and the results identified differentially expressed genes related to muscle development, adipogenesis, amino acid metabolism, fatty acid metabolism and inosine synthesis. In conclusion, the study identified functional genes, elucidated the mechanisms associated with carcass quality traits, meat quality traits and other related traits, and provided means of genetic enhancement to improve meat quality traits and carcass traits in Chinese commercial pigs.

An efficient protection-free chemical synthesis of inosine 5'-nucleotides

A facile, straightforward, reliable, and efficient chemical synthesis of inosine nucleotides such as inosine-5′-monophosphate, inosine-5′-diphosphate, and inosine-5′-triphosphate, starting from inosine is delineated. The inosine-5′-monophosphate is achieved by the highly regioselective monophosphorylation of inosine using the Yoshikawa procedure. The inosine-5′-diphosphate is obtained by the coupling reaction of tributylammonium phosphate with an activated inosine-5′-monophosphate using zinc chloride as a catalyst. The inosine-5′-triphosphate is efficiently achieved by the improved “one-pot, three-step” Ludwig synthetic strategy. In all the cases, the resulting final product is isolated in good yields with high purity (>99.5%).

Hypoxic Vasospasm Mediated by cIMP: When Soluble Guanylyl Cyclase Turns Bad.

:In a number of isolated blood vessel types, hypoxia causes an acute contraction that is dependent on the presence of nitric oxide and activation of soluble guanylyl cyclase. It is more pronounced when the preparations are constricted and is therefore termed hypoxic augmentation of vasoconstriction. This hypoxic response is accompanied by increases in the intracellular level of inosine 5′-triphosphate and in the synthesis of inosine 3′,5′-cyclic monophosphate (cIMP) by soluble guanylyl cyclase. The administration of exogenous cIMP or inosine 5′-triphosphate causes augmented vasoconstriction to hypoxia. Furthermore, the vasoconstriction evoked by hypoxia and cIMP is associated with increased activity of Rho kinase (ROCK), indicating that cIMP may mediate the hypoxic effect by sensitizing the myofilaments to Ca2+ through ROCK. Hypoxia is implicated in exaggerated vasoconstriction in the pathogenesis of coronary artery disease, myocardial infarction, hypertension, and stroke. The newly found role of cIMP may help to identify unique therapeutic targets for certain cardiovascular disorders.

Synthesis of Inosine Polyphosphates from Inosine 5’-Phosphate

Treatment of inosine 5’-phosphate (IMP) with 2,2’-dithiodianiline/triphenylphosphine provided an efficient method for the synthesis of inosine 5′-phosphoropiperidate under mild conditions. Inosine 5’-triphosphate (ITP) and inosine 5′-diphosphate (IDP) were efficiently synthesized from inosine 5’-phosphoropiperidate via the activation P(V)—N bond by 4,5-dicyanoimidazole.

In and out: an analysis of epibiotic vs periplasmic bacterial predators.

Bdellovibrio and like organisms (BALO) are obligate predators of Gram-negative bacteria, belonging to the α- and δ-proteobacteria. BALO prey using either a periplasmic or an epibiotic predatory strategy, but the genetic background underlying these phenotypes is not known. Here we compare the epibiotic Bdellovibrio exovorus and Micavibrio aeruginosavorus to the periplasmic B. bacteriovorus and Bacteriovorax marinus. Electron microscopy showed that M. aeruginosavorus, but not B. exovorus, can attach to prey cells in a non-polar manner through its longitudinal side. Both these predators were resistant to a surprisingly high number of antibiotic compounds, possibly via 26 and 19 antibiotic-resistance genes, respectively, most of them encoding efflux pumps. Comparative genomic analysis of all the BALOs revealed that epibiotic predators have a much smaller genome (ca. 2.5 Mbp) than the periplasmic predators (ca. 3.5 Mbp). Additionally, periplasmic predators have, on average, 888 more proteins, at least 60% more peptidases, and one more rRNA operon. Fifteen and 219 protein families were specific to the epibiotic and the periplasmic predators, respectively, the latter clearly forming the core of the periplasmic 'predatome', which is upregulated during the growth phase. Metabolic deficiencies of epibiotic genomes include the synthesis of inosine, riboflavin, vitamin B6 and the siderophore aerobactin. The phylogeny of the epibiotic predators suggests that they evolved by convergent evolution, with M. aeruginosavorus originating from a non-predatory ancestor while B. exovorus evolved from periplasmic predators by gene loss.

Two‐Step, One‐Pot Synthesis of Inosine, Guanosine, and 2′‐Deoxyguanosine O6‐Ethers via Intermediate O6‐(Benzotriazol‐1‐yl) Derivatives

A simple method for the etherification at the O(6)-position of silyl-protected inosine, guanosine, and 2'-deoxyguanosine is described. Typically, a THF solution of the silylated nucleoside is treated with 1H-benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) and Cs(2)CO(3) under a nitrogen atmosphere. Conversion to the O(6)-(benzotriazol-1-yl) ethers occurs within about 10 min for inosine, and within about 60 min for guanosine and 2'-deoxyguanosine. Then, for reaction with alcohols, the reaction mixture is evaporated and the O(6)-(benzotriazol-1-yl) ether is treated with Cs(2)CO(3) and an appropriate alcohol, at room temperature. On the other hand, for reaction with phenols, Cs(2)CO(3) and the appropriate phenol are added to the reaction mixture without evaporation, and the reaction is carried out at 70°C. Subsequently, workup, isolation, and purification lead to the requisite O(6)-alkyl or O(6)-aryl ethers in good to excellent yields.

Evidence of a new phosphoryl transfer system in nucleotide metabolism

Crude rat liver extract showed AMP-AMP phosphotransferase activity which, on purification, was ascribed to a novel interaction between adenylate kinase, also known as myokinase (EC 2.7.4.3), and adenosine kinase (EC 2.7.1.20). The activity was duplicated using the same enzymes purified from recombinant sources. The reaction requires physical contact between myokinase and adenosine kinase, and the net reaction is aided by the presence of adenosine deaminase (EC 3.5.4.4), which fills the gap in the energy balance of the phosphoryl transfer and shifts the equilibrium towards ADP and inosine synthesis. The proposed mechanism involves the association of adenosine kinase and myokinase through non-covalent, transient interactions that induce slight conformational changes in the active site of myokinase, bringing two already bound molecules of AMP together for phosphoryl transfer to form ADP. The proposed mechanism suggests a physiological role for the enzymes and for the AMP-AMP phosphotransferase reaction under conditions of extreme energy drain (such as hypoxia or temporary anoxia, as in cancer tissues) when the enzymes cannot display their conventional activity because of substrate deficiency.

Synthesis of nucleosidic bonds using a nucleoside hydrolase in aqueous-organic media

The synthesis of inosine from nonactivated and nonprotected ribose and hypoxanthine was performed by enzyme-catalyzed condensation, using a nucleoside hydrolase from Crithidia fasciculata expressed in Escherichia coli with a synthetic gene. One round of directed evolution was performed in the presence of dimethylformamide, used to lower the water activity in the reaction media, leading to a double mutant (Asp54Asn, Arg137Gly). It afforded a doubling of the specificity constant for the hydrolysis of p-nitrophenol-β-d-riboside in the presence of 40% acetonitrile. In aqueous conditions, concentrations of 0.1 and 0.2 mM inosine were obtained, starting from 25 mM hypoxanthine and 2 or 3 M ribose, respectively. With 20% acetonitrile increases of 95% and 60% were observed. These conversions are very low, and the work exemplifies the difficulties encountered when trying to define conditions for hydrolase-catalyzed condensation and at the same time evolve an enzyme to perform well in these a priori unknown conditions.

Enhancement of inosine production by Bacillus subtilis through suppression of carbon overflow by sodium citrate

Sodium citrate, added to culture medium at 2 g l-1, increased inosine production by B. subtilis by 18% to 16 g l-1. The phosphoenolpyruvate (PEP) pool was increased by 2.3-fold, while the pyruvate and acetate pools were decreased by 78% and 57%, respectively. Pyruvate kinase might thus be a regulatory site for inosine synthesis.

Correlation between key enzyme activities in the inosine synthetic pathway and inosine production

The specific activities of key enzymes relating to the pathway of inosine synthesis of three different bacterial strains including high-yield, low-yield and wild strains were determined and compared systematically. A close relationship between inosine production and the specific activities of key enzymes was found. According to the enzyme characteristics of the high-yield strain, suggestions on further strain improvement by genetic engineering were proposed. An enzymology study is suggested as an effective way to facilitate the screening of high-yield strain.

Correlation between key enzyme activities in the inosine synthetic pathway and inosine production

The specific activities of key enzymes relating to the pathway of inosine synthesis of three different bacterial strains including high-yield, low-yield and wild strains were determined and compared systematically. A close relationship between inosine production and the specific activities of key enzymes was found. According to the enzyme characteristics of the high-yield strain, suggestions on further strain improvement by genetic engineering were proposed. An enzymology study is suggested as an effective way to facilitate the screening of high-yield strain.

The Kinetic Mechanism of the Human Bifunctional Enzyme ATIC (5-Amino-4-imidazolecarboxamide Ribonucleotide Transformylase/Inosine 5′-Monophosphate Cyclohydrolase): A SURPRISING LACK OF SUBSTRATE CHANNELING

5-Amino-4-imidazolecarboxamide ribonucleotide transformylase/IMP cyclohydrolase (ATIC) is a bifunctional protein possessing two enzymatic activities that sequentially catalyze the last two steps in the pathway for de novo synthesis of inosine 5'-monophosphate. This bifunctional enzyme is of particular interest because of its potential as a chemotherapeutic target. Furthermore, these two catalytic activities reside on the same protein throughout all of nature, raising the question of whether there is some kinetic advantage to the bifunctionality. Rapid chemical quench, stopped-flow absorbance, and steady-state kinetic techniques were used to elucidate the complete kinetic mechanism of human ATIC. The kinetic simulation program KINSIM was used to model the kinetic data obtained in this study. The detailed kinetic analysis, in combination with kinetic simulations, provided the following key features of the enzyme reaction pathway. 1) The rate-limiting step in the overall reaction (2.9 +/- 0.4 s(-1)) is likely the release of tetrahydrofolate from the formyltransferase active site or a conformational change associated with tetrahydrofolate release. 2) The rate of the reverse transformylase reaction (6.7 s(-1)) is approximately 2-3-fold faster than the forward rate (2.9 s(-1)), whereas the cyclohydrolase reaction is essentially unidirectional in the forward sense. The cyclohydrolase reaction thus draws the overall bifunctional reaction toward the production of inosine monophosphate. 3) There was no kinetic evidence of substrate channeling of the intermediate, the formylaminoimidazole carboxamide ribonucleotide, between the formyltransferase and the cyclohydrolase active sites.

Experimental proof for the regulation ofSalmonella typhimurium purB bypurR

Salmonella typhimurium purB encodes adenylosuccinate lyase (ASL), the enzyme that catalyzes step 8 in the pathway forde novo synthesis of inosine 5′-monophosphate (IMP) and also the final reaction in the two-step sequence from IMP to adenosine monophosphate (AMP). The nucleotide sequence ofpurB was obtained by the genetic map and sequence homologous analysis. The conservedpur operator inpurB was identified to be located 185 bp downstream of the initiation codon and overlaps codons 62 – 67 in the protein-coding region. The binding of PurR to this operator was demonstrated by gel retardation experiment and site-directed mutagenesis, indicating that thepurB is under the control ofpurR. We also answered why previous study had conflicting report concerning the regulation ofpurB bypurR by identifying the junction site ofpurB tolacZ in apurB- MudJ (lacZ,Kan r) fusion strain. This result strongly supports that thepurB is the second gene in theycfC-purB operon.

An Improved Synthesis of Inosine 3′-Phosphoramidite

A protocol for the efficient conversion of inosine into its 3′-phosphoramidite synthon for solid phase oligonucleotide synthesis has been developed.

Purine salvage pathways in the Australian termite, Nasutitermes walkeri hill

Extracts of the degutted body of the termite Nasutitermes walkeri contained adenine phosphoribosyltransferase (APRT) (0.277 ± 0.014 nmol/min/mg protein) but neither hypoxanthine-guanine phosphoribosyltransferase (HGPRT) nor inosine kinase indicating that the synthesis of inosine is the committed step in the catabolism of IMP and AMP to urate. The presence of a low activity of adenosine kinase (0.025 ± 0.002 nmol/min/mg protein) may be required to prevent depletion of the adenylate pool. Bacterial extracts from the gut by contrast contained the full range of salvage enzymes including HGPRT (0.117 ± 0.003 nmol/min/mg protein), APRT (0.371 ± 0.015 nmol/min/mg protein), adenosine kinase (0.068 ± 0.025 nmol/min/mg protein) and inosine kinase (0.032 ± 0.002 nmol/min/mg protein). The inability of N. walkeri to salvage inosine and hypoxanthine suggests a mechanism for the apparently uncontrolled synthesis of urate, by termites, during laboratory storage.

Pigeon liver purine nucleoside phosphorylase characterization: kinetic behaviour and sulfhydryl groups

1. 1. Monomeric pigeon liver purine nucleoside phosphorylase did not exhibit the phenomenon of substrate activation at high substrate concentrations. 2. 2. Initial velocity studies and product inhibition patterns for the synthesis of inosine were consistent with a Random Bi, Bi mechanism. 3. 3. One mole of native purine nucleoside phosphorylase binds with 2 mol of 5,5′-dithiobis-(2-nitro-benzoic acid) and there is an increase in number upon denaturation with sodium dodecyl sulfate. 4. 4. Hypoxanthine, inosine, orthophosphate and ribose-1-phosphate partially protect the sulfhydryl groups from reaction with 5,5′-dithiobis-(2-nitrobenzoic acid). 5. 5. p-Chloromercuribenzoate and 5,5′-dithiobis-(2-nitrobenzoic acid) inhibit the activity of the pigeon liver enzyme according to a second order reaction kinetics.

Inosine synthesis and phosphoribosylpyrophosphate availability in rat liver cells in the presence of allopurinol.

In the presence of allopurinol, apparent phosphoribosylpyrophosphate (PP-ribose-P) availability as measured by adenine incorporation into ribonucleotides was decreased in rat liver cells, hypoxanthine incorporation into ribonucleotides was increased, and there was a large synthesis of inosine from hypoxanthine. Inosine was formed directly by the reversal of the purine nucleoside phosphorylase reaction which was very rapid in liver cells. We tested the hypothesis that utilization of ribose 1-phosphate for inosine synthesis could decrease PP-ribose-P availability. Our results indicate that the apparent decrease of PP-ribose-P availability in the presence of allopurinol was due to competition between adenine and hypoxanthine salvage pathways into nucleotides, and not to the synthesis of inosine.

Utilization of 2,6-diaminopurine by Salmonella typhimurium.

The pathway for the utilization of 2,6-diaminopurine (DAP) as an exogenous purine source in Salmonella typhimurium was examined. In strains able to use DAP as a purine source, mutant derivatives lacking either purine nucleoside phosphorylase or adenosine deaminase activity lost the ability to do so. The implied pathway of DAP utilization was via its conversion to DAP ribonucleoside by purine nucleoside phosphorylase, followed by deamination to guanosine by adenosine deaminase. Guanosine can then enter the established purine salvage pathways. In the course of defining this pathway, purine auxotrophs able to utilize DAP as sole purine source were isolated and partially characterized. These mutants fell into several classes, including (i) strains that only required an exogenous source of guanine nucleotides (e.g., guaA and guaB strains); (ii) strains that had a purF genetic lesion (i.e., were defective in alpha-5-phosphoribosyl 1-pyrophosphate amidotransferase activity); and (iii) strains that had constitutive levels of purine nucleoside phosphorylase. Selection among purine auxotrophs blocked in the de novo synthesis of inosine 5'-monophosphate, for efficient growth on DAP as sole source of purine nucleotides, readily yielded mutants which were defective in the regulation of their deoxyribonucleoside-catabolizing enzymes (e.g., deoR mutants).

Lesch-Nyhan syndrome: the synthesis of inosine 5'-phosphate in the hypoxanthine-guanine phosphoribosyltransferase-deficient erythrocyte by alternate biochemical pathways.

Erythrocytes, obtained from a normal adult male and from a patient with Lesch-Nyhan syndrome, were incubated with [8-14C]adenine and [8-14C]hypoxanthine (Table 1). The labeled adenine was utilized to about the same extent for the synthesis of AMP by the normal subject's and the patient's erythrocytes. Deamination of AMP to IMP occurred to about the same extent in both samples. In contrast, hypoxanthine was utilized extensively for IMP synthesis in the normal erythrocyte only. The amount of total label in the IMP was about 100 times that of the Lesch-Nyhan erythrocyte, a consequence of the deficiency of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity in the syndrome. No significant labeling of the AMP occurred. When aliquots of erythrocytes from both sources were incubated with 4-amino-5-imidazolecarboxamide (AICA) and sodium [14C]formate, extensive labeling of the IMP occurred in normal and in Lesch-Nyhan erythrocytes. The data suggest that AICA serves as a substrate for the adenine phosphoribosyltransferase (APRT) of the Lesch-Nyhan erythrocyte and that the ribotide of AICA, 5'-phosphoribosyl-5-aminoimidazole-4-carboxamide (AICAR), undergoes formylation by labeled N10-formyl tetrahydrofolic acid formed from the reaction of sodium [14C]formate with the tetrahydrofolic acid of the cell. The formyl-AICAR undergoes ring closure to IMP by a series of reactions comparable to those described for the normal erythrocyte. When 5-amino-1-ribosyl-4-imidazolecarboxamide (rAICA) and sodium [14C]formate were incubated with erythrocyte suspensions, extensive utilization for IMP synthesis was also observed in normal erythrocytes and in erythrocytes from Lesch-Nyhan patients (Table 2). The reaction sequence is somewhat different from that of AICA. AICA is not a substrate for the purine nucleoside phosphorylase of rabbit or human erythrocytes. The mechanism of rAICA utilization is visualized as a direct phosphorylation of the ribosyl compound, possibly by the adenosine kinase of the human cell. The ribotide, AICAR, formed by this mechanism, undergoes formylation and ring closure, yielding IMP. The glutamine antagonist, diazooxonorleucine (DON), was added to aliquots of patients' cells incubated with rAICA and sodium [14C]formate. DON is an effective inhibitor of the conversion of IMP to GMP and its presence in an incubation suspension resulted in a somewhat greater radioactivity of the total cellular IMP. The extension of the current studies to Lesch-Nyhan cells in culture may serve to assist in the direct evaluation of the regulatory role of IMP in the de novo pathway of purine nucleotide biosynthesis. Because of the substrate requirements of the reactions, the metabolism of AICA and rAICA may also serve to differentiate the roles of purine nucleotides and of phosphoribosylpyrophosphate (PRPP) in the pathway regulation. The findings presented also offer a possible therapeutic approach to the early treatment of the disease in the afflicted neonate...