Enzymatic synthesis of novel purine nucleosides bearing a chiral benzoxazine fragment.

A series of ribo- and deoxyribonucleosides bearing 2-aminopurine as a nucleobase with 7,8-difluoro- 3,4-dihydro-3-methyl-2H-[1,4]benzoxazine (conjugated directly or through an aminohexanoyl spacer) was synthesized using an enzymatic transglycosylation reaction. Nucleosides 3-6 were resistant to deamination under action of adenosine deaminase (ADA) Escherichia coli and ADA from calf intestine. The antiviral activity of the modified nucleosides was evaluated against herpes simplex virus type 1 (HSV-1, strain L2). It has been shown that at sub-toxic concentrations, nucleoside (S)-4-[2-amino-9-(ß-D-ribofuranosyl)-purin-6-yl]-7,8-difluoro-3,4-dihydro-3-methyl-2H-[1,4]benzoxazine exhibit significant antiviral activity (SI > 32) on the model of HSV-1 in vitro, including an acyclovir-resistant virus strain (HSV-1, strain L2/R).

Coordinated Biosynthesis of the Purine Nucleoside Antibiotics Aristeromycin and Coformycin in Actinomycetes.

Purine nucleoside antibiotic pairs, concomitantly produced by a single strain, are an important group of microbial natural products. Here, we report a target-directed genome mining approach to elucidate the biosynthesis of the purine nucleoside antibiotic pair aristeromycin (ARM) and coformycin (COF) in Micromonospora haikouensis DSM 45626 (a new producer for ARM and COF) and Streptomyces citricolor NBRC 13005 (a new COF producer). We also provide biochemical data that MacI and MacT function as unusual phosphorylases, catalyzing an irreversible reaction for the tailoring assembly of neplanocin A (NEP-A) and ARM. Moreover, we demonstrate that MacQ is shown to be an adenosine-specific deaminase, likely relieving the potential "excess adenosine" for producing cells. Finally, we report that MacR, an annotated IMP dehydrogenase, is actually an NADPH-dependent GMP reductase, which potentially plays a salvage role for the efficient supply of the precursor pool. Hence, these findings illustrate a fine-tuned pathway for the biosynthesis of ARM and also open the way for the rational search for purine antibiotic pairs.IMPORTANCE ARM and COF are well known for their prominent biological activities and unusual chemical structures; however, the logic of their biosynthesis has long been poorly understood. Actually, the new insights into the ARM and COF pathway will not only enrich the biochemical repertoire for interesting enzymatic reactions but may also lay a solid foundation for the combinatorial biosynthesis of this group of antibiotics via a target-directed genome mining strategy.

d-Sedoheptulose-7-phosphate is a common precursor for the heptoses of septacidin and hygromycin B.

Seven-carbon-chain-containing sugars exist in several groups of important bacterial natural products. Septacidin represents a group of l-heptopyranoses containing nucleoside antibiotics with antitumor, antifungal, and pain-relief activities. Hygromycin B, an aminoglycoside anthelmintic agent used in swine and poultry farming, represents a group of d-heptopyranoses-containing antibiotics. To date, very little is known about the biosynthesis of these compounds. Here we sequenced the genome of the septacidin producer and identified the septacidin gene cluster by heterologous expression. After determining the boundaries of the septacidin gene cluster, we studied septacidin biosynthesis by in vivo and in vitro experiments and discovered that SepB, SepL, and SepC can convert d-sedoheptulose-7-phosphate (S-7-P) to ADP-l-glycero-ß-d-manno-heptose, exemplifying the involvement of ADP-sugar in microbial natural product biosynthesis. Interestingly, septacidin, a secondary metabolite from a gram-positive bacterium, shares the same ADP-heptose biosynthesis pathway with the gram-negative bacterium LPS. In addition, two acyltransferase-encoding genes sepD and sepH, were proposed to be involved in septacidin side-chain formation according to the intermediates accumulated in their mutants. In hygromycin B biosynthesis, an isomerase HygP can recognize S-7-P and convert it to ADP-d-glycero-ß-d-altro-heptose together with GmhA and HldE, two enzymes from the Escherichia coli LPS heptose biosynthetic pathway, suggesting that the d-heptopyranose moiety of hygromycin B is also derived from S-7-P. Unlike the other S-7-P isomerases, HygP catalyzes consecutive isomerizations and controls the stereochemistry of both C2 and C3 positions.

Identification and Interrogation of the Herbicidin Biosynthetic Gene Cluster: First Insight into the Biosynthesis of a Rare Undecose Nucleoside Antibiotic.

Herbicidins are adenosine-based nucleoside antibiotics with an unusual tricyclic undecose core decorated with a (5-hydroxy)tiglyl moiety. Feeding studies are herein reported demonstrating that the tricyclic core is derived from d-glucose and d-ribose, whereas the tiglyl moiety is derived from an intermediate of l-isoleucine catabolism. Identification of the gene cluster for herbicidin A biosynthesis in Streptomyces sp. L-9-10 as well as its verification by heterologous expression in a nonproducing host are described, and the results of in vitro characterization of a carboxyl methyltransferase encoded in the cluster, Her8, are presented. Based on these observations, a biosynthetic pathway is proposed for herbicidins.

Biosynthesis of 4-aminoheptose 2-epimers, core structural components of the septacidins and spicamycins.

Septacidins and spicamycins are acylated 4-aminoheptosyl-ß-N-glycosides produced by Streptomyces fimbriatus and S. alanosinicus, respectively. Their structures are highly conserved, but differ in the stereochemistry of the 4-aminoheptosyl residues. The origin of this stereochemistry is unknown, but is presumably because of the difference in their biosynthetic pathways. We have synthesized the septacidin 4-aminoheptose to verify the difference between septacidin and spicamycin. Isotopic enrichment studies were undertaken using S. fimbriatus, and show that the septacidin heptose is derived from the pentose phosphate pathway. This indicates conserved pathways leading to the biosynthesis of 4-amino-4-deoxy-L-gluco-heptose or 4-amino-4-deoxy-L-manno-heptose.

A purine nucleotide biosynthesis enzyme guanosine monophosphate reductase is a suppressor of melanoma invasion.

Melanoma is one of the most aggressive types of human cancers, and the mechanisms underlying melanoma invasive phenotype are not completely understood. Here, we report that expression of guanosine monophosphate reductase (GMPR), an enzyme involved in de novo biosynthesis of purine nucleotides, was downregulated in the invasive stages of human melanoma. Loss- and gain-of-function experiments revealed that GMPR downregulates the amounts of several GTP-bound (active) Rho-GTPases and suppresses the ability of melanoma cells to form invadopodia, degrade extracellular matrix, invade in vitro, and grow as tumor xenografts in vivo. Mechanistically, we demonstrated that GMPR partially depletes intracellular GTP pools. Pharmacological inhibition of de novo GTP biosynthesis suppressed whereas addition of exogenous guanosine increased invasion of melanoma cells as well as cells from other cancer types. Our data identify GMPR as a melanoma invasion suppressor and establish a link between guanosine metabolism and Rho-GTPase-dependent melanoma cell invasion.

Modeling cellular compartmentation in one-carbon metabolism.

Folate-mediated one-carbon metabolism (FOCM) is associated with risk for numerous pathological states including birth defects, cancers, and chronic diseases. Although the enzymes that constitute the biological pathways have been well described and their interdependency through the shared use of folate cofactors appreciated, the biological mechanisms underlying disease etiologies remain elusive. The FOCM network is highly sensitive to nutritional status of several B-vitamins and numerous penetrant gene variants that alter network outputs, but current computational approaches do not fully capture the dynamics and stochastic noise of the system. Combining the stochastic approach with a rule-based representation will help model the intrinsic noise displayed by FOCM, address the limited flexibility of standard simulation methods for coarse-graining the FOCM-associated biochemical processes, and manage the combinatorial complexity emerging from reactions within FOCM that would otherwise be intractable.

Thermus thermophilus strains active in purine nucleoside synthesis.

Several strains of Thermus thermophilus were tested in order to detect purine nucleoside synthase activity using pyrimidine nucleosides as the sugar-donor and adenine or hypoxanthine as bases. High productivity values (t =1 hr) were obtained while completely avoiding adenosine-deaminase degradation of the products. N-2-deoxy-ribosyltransferase activity is described for the first time in hyperthermophilic bacteria.

Anicemycin, a new inhibitor of anchorage-independent growth of tumor cells from Streptomyces sp. TP-A0648.

The anchorage-independence of cells is closely related to their tumorigenicity. In the screening of inhibitors of anchorage-independent growth of tumor cells, anicemycin was isolated from the fermentation broth of an actinomycete strain TP-A0648. The producing strain was isolated from a leaf of Aucuba japonica collected in Toyama, Japan and identified as Streptomyces sp. based on the taxonomic data. The structure of anicemycin was elucidated as a new analog of spicamycin by NMR and MS analysis. Anicemycin inhibited the anchorage-independent growth of the human ovary cancer SKOV-3 cells with an IC50 of 0.015 microM about three times more potently than their anchorage-dependent growth.

A new stable isotope method enables the simultaneous measurement of nucleic acid and protein synthesis in vivo in mice.

We developed a method based on the incorporation of 13C2-units derived from [U-13C]glycine that allows the simultaneous quantification of tissue protein and RNA synthesis in vivo. Two groups of 26 mice were fed diets containing a high (HF diet) or a low quantity of fiber (LF diet). After 6 d, [U13C]glycine was added to the diet and groups of four mice were killed after 2, 4, 6, 8, 12 and 24 h. Hepatic and intestinal mucosal free and RNA-bound purine nucleosides were extracted and enzymically degraded to allantoin. Allantoin was degraded to glyoxylate, which was then reductively aminated to glycine, which contains the two 13C-atoms incorporated via de novo synthesis. Ingestion of the HF diet was associated with significantly (P < 0.05) higher rates of total RNA synthesis in both the liver ( HF diet, 29%/d; LF diet, 21%/d) and mucosa (HF diet, 27%/d; LF diet, 17 %/d). The mean rates of RNA synthesis in each tissue were significantly (P < 0.01) lower than the respective rates of protein synthesis (liver, 67%/d; mucosa, 74%/d). The isotopic enrichment of the free purine nucleotide pool increased rapidly and exponentially, but the steady-state value was substantially (P < 0. 001) lower than that of the RNA-bound purines. The results suggest that the free nucleotide pool consists of two kinetically distinct compartments, one of which is small and has a rapid rate of turnover. This, we propose, acts as the RNA precursor pool. The other is large, has a low rate of turnover and, we believe, is the pool of adenosine triphosphate involved in cellular energetics.

Structure-antitumor activity relationship of semi-synthetic Spicamycin derivatives.

New derivatives of spicamycin modified at the fatty acid moieties of the molecule were synthesized and their structure-activity relationships were examined. The antitumor activity was greatly influenced by modification of the fatty acid moieties to tetradecadienoyl or dodecadienoyl analogues exhibiting better antitumor activity against COL-1 human colon cancer xenograft than SPM VIII.

Enzymatic synthesis of purine 2'-deoxyriboside and its properties as an inhibitor of adenosine deaminases from calf intestinal mucosa and Bacillus cereus.

Enzymatic synthesis of purine 2'-deoxyriboside was obtained by reacting purine with excess 2-deoxy-alpha-D-ribose-1-phosphate in the presence of commercial bovine nucleoside phosphorylase; the product was isolated by semipreparative reverse phase HPLC with an overall 62% yield. Purine 2'-deoxyriboside was shown to behave as a competitive inhibitor of adenosine deaminase from calf intestinal mucosa and Bacillus cereus, with apparent Ki values of 4.5 and 8.5 microM, respectively.

Pentatrichomonas hominis: purine salvage pathway.

1. Pentatrichomonas hominis was found incapable of de novo synthesis of purines. 2. Pentatrichomonas hominis can salvage adenine, guanine, hypoxanthine, adenosine, guanosine and inosine, but not xanthine for the synthesis of nucleotides. 3. HPLC tracing of radiolabelled purines or purine nucleosides revealed that adenine, adenosine and hypoxanthine are incorporated into adenine nucleotides and IMP through a similar channel while guanine and guanosine are salvaged into guanine nucleotides via another route. There appears to be no direct interconversion between adenine and guanine nucleotides. Interconversion between AMP and IMP was observed. 4. Assays of purine salvage enzymes revealed that P. hominis possess adenosine kinase; adenosine, guanosine and inosine phosphotransferases; adenosine, guanosine and inosine phosphorylases and AMP deaminase.

C(2')-substituted purine nucleoside analogs. Interactions with adenosine deaminase and purine nucleoside phosphorylase and formation of analog nucleotides.

Four C(2')-substituted 2'-deoxyadenosines were examined as substrates for human erythrocytic adenosine deaminase and for formation of intracellular nucleotide analogs in human erythrocytes, lymphocytes and murine Sarcoma 180 cells: 9-(2'-deoxy-2'-fluoro-beta-D-ribofuranosyl)adenine, 9-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)adenine, 9-(2'-azido-2'-deoxy-beta-D-ribofuranosyl)adenine (2'-N3-riboA) and 9-(2-azido-2'-deoxy-beta-D-arabinofuranosyl)adenine. All four adenosine analogs were substrates of human erythrocytic adenosine deaminase, but the corresponding inosine analogs (synthesized by the adenosine deaminase reaction) were highly resistant to cleavage by human erythrocytic purine nucleoside phosphorylase. Only 9-(2'-deoxy-2'-fluoro-beta-D-ribofuranosyl)hypoxanthine underwent very slow phosphorolysis, and no inhibition of inosine phosphorolysis was detected when a 30 microM concentration of any studied inosine analog was added to a reaction mixture containing 30 microM inosine (the Km concentration). Kinetic parameters were determined for the deamination of the adenosine analogs. The greatest affinity for adenosine deaminase was found with 2'-N3-ribo A (Ki = 2 microM), but the reaction velocity was highest with the F-substituted analogs. All four adenosine analogs formed triphosphate nucleotides after incubation with human erythrocytes, murine Sarcoma 180 cells, or human lymphocytes (tested only with the F analogs) in the presence of deoxycoformycin.

Theophylline inhibition of renal and cerebral nucleoside formation.

1 Theophylline inhibits the enzymatic formation of purine nucleosides, among these adenosine (dephosphorylated adenosine 5'-monophosphate), in kidney and brain of the rat. 2 Some pharmacological effects of theophylline on regional blood flow and electrophysiological activity of the nervous system may be caused by inhibition of the endogenous formation of adenosine.

Elucidation of aberrant purine metabolism: application to hypoxanthine-guanine phosphoribosylstransferase- and adenosine kinase-deficient mutants, and IMP dehydrogenase- and adenosine deaminase-inhibited human lymphoblasts.

We propose that the ratio of [14C]formate-labelled purine nucleosides and bases (both intra and extracellular) to nucleic acid purines provides, in exponentially growing cultures, a sensitive index for comparative studies of purine metabolism. This ratio was 4-fold greater for an HGPRT- mutant than for the parental HGPRT+ human lymphoblast line. The major components of the labelled nucleoside and base fraction were hypoxanthine and inosine. By blocking adenosine deaminase activity with coformycin we found that approx. 90% of inosine was formed directly from IMP rather than the route IMP leads to AMP leads to adenosine leads to inosine. The ratio of labelled base + nucleosides to nucleic acids was essentially unchagned for an AK- lymphoblast line and 2-fold greater than control for an HGPRT(-)-KAK- line, demonstrating that a deficiency of adenosine kinase alone has little effect on the accumulation of purine nucleosides and bases. Although adenosine was a minor component of the nucleoside and base fraction, the adenosine fraction increased from 3 to 13% with the addition of coformycin to the HGPRT(-)-AK- line. In the parental and HGPRT- lines, adenosine was shown to be primarily phosphorylated rather than deaminated at concentrations less than 5 microM. Inhibition of IMP dehydrogenase activity by mycophenolic acid caused a 12- and 3-fold increase in the rate of production of labelled base and nucleoside in the parent and HGPRT- cells respectively. These results suggest that a mutationally induced partial deficiency in the activities converting IMP to guanine nucleotides may result in an increased catabolism of IMP.

Isolation and characterization of spontaneously arising auxotrophic and Kanagawa phenomenon-negative mutants of Vibrio parahaemolyticus.

As a first step toward developing a system of genetic exchange between Vibrio parahaemolyticus strains, spontaneously arising auxotrophic and Kanagawa phenomenon-negative (KP-) mutants were isolated and characterized. Auxotrophic mutants were selected by nalidixic acid enrichment of parental cultures. Some Cys- and Arg- mutants of a KP+ strain were found to be KP-. Reversion to prototrophy by these strains was not accompanied by a return to the parental KP+ phenotype. Additionally, two prototrophic KP- mutants were isolated. No detectable levels of vibriolysin were found in supernatant extracts of KP- mutants by slide gel immunodiffusion analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, or assay for lethal activity in mice. All Cys-, Arg-, and Pur- mutants tested reverted to a different auxotrophy (phenotypic interconversion) as well as to prototrophy. The possible role of insertion sequence-like elements in vibriolysin production and phenotypic interconversion is discussed.