Characterization of SepE and SepF for the N6-Glycosylated Adenine Structure Formation in Septacidin Biosynthesis.

Septacidin (1) represents a group of nucleoside antibiotics possessing a unique N6-glycosylated adenine core. They exhibit some fascinating bioactivities that are rare for other nucleoside antibiotics. Here we demonstrate that this unique structure in septacidin is formed by SepE and SepF. SepE is an unprecedented Fe(II)-dependent glycosyltransferase decorating the N6-position of AMP using ADP-l-glycero-ß-d-manno-heptose (6) as a sugar donor. The Fe(II) may help SepE to bind AMP. SepF is an unusual glycosidase that detaches the N9-ribosyl-5-phosphate.

Elucidation of the Herbicidin Tailoring Pathway Offers Insights into Its Structural Diversity.

The biosynthetic gene clusters for herbicidins ( hbc) and aureonuclemycin ( anm) were identified in Streptomyces sp. KIB-027 and Streptomyces aureus, respectively. The roles of genes possibly involved in post-core-assembly steps in herbicidin biosynthesis in these clusters and a related her cluster were studied. Through systematic gene deletions, structural elucidation of the accumulated intermediates in the mutants, and in vitro verification of the encoded enzymes, the peripheral modification pathway for herbicidin biosynthesis is now fully established.

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.

Oxidative stress increases M1dG, a major peroxidation-derived DNA adduct, in mitochondrial DNA.

Reactive oxygen species (ROS) are formed in mitochondria during electron transport and energy generation. Elevated levels of ROS lead to increased amounts of mitochondrial DNA (mtDNA) damage. We report that levels of M1dG, a major endogenous peroxidation-derived DNA adduct, are 50-100-fold higher in mtDNA than in nuclear DNA in several different human cell lines. Treatment of cells with agents that either increase or decrease mitochondrial superoxide levels leads to increased or decreased levels of M1dG in mtDNA, respectively. Sequence analysis of adducted mtDNA suggests that M1dG residues are randomly distributed throughout the mitochondrial genome. Basal levels of M1dG in mtDNA from pulmonary microvascular endothelial cells (PMVECs) from transgenic bone morphogenetic protein receptor 2 mutant mice (BMPR2R899X) (four adducts per 106 dG) are twice as high as adduct levels in wild-type cells. A similar increase was observed in mtDNA from heterozygous null (BMPR2+/-) compared to wild-type PMVECs. Pulmonary arterial hypertension is observed in the presence of BMPR2 signaling disruptions, which are also associated with mitochondrial dysfunction and oxidant injury to endothelial tissue. Persistence of M1dG adducts in mtDNA could have implications for mutagenesis and mitochondrial gene expression, thereby contributing to the role of mitochondrial dysfunction in diseases.

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.

Optimization of Herbicidin A Production in Submerged Culture of Streptomyces scopuliridis M40.

Herbicidin A is a potent herbicide against dicotyledonous plants as well as an antibiotic against phytopathogens. In this study, fermentation parameters for herbicidin A production in submerged culture of Streptomyces scopuliridis M40 were investigated. The herbicidin A concentration varied with the C/N ratio. High C/N ratios (>4) resulted in a herbicidin A production of more than 900 mg/l, whereas maximally 600 mg/l was obtained at ratios between 1 and 3.5. In 5-L batch fermentation, there was a positive correlation between the oxygen uptake rate (OUR) and herbicidin A production. Once the OUR increased, the substrate consumption rate increased, leading to an increase in volumetric productivity. Mechanical shear force affected the hyphal morphology and OUR. When the medium value of hyphal size ranged from 150 to 180 µm, high volumetric production of herbicidin A was obtained with OUR values >137 mg O2/l·h. The highest herbicidin A concentration of 956.6 mg/l was obtained at 500 rpm, and coincided with the highest relative abundance of hyphae of 100-200 µm length and the highest OUR during cultivation. Based on a constant impeller tip speed, which affects hyphal morphology, herbicidin A production was successfully scaled up from a 5-L jar to a 500-L pilot vessel.

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.

Biosynthesis of anti-HCV compounds using thermophilic microorganisms.

This work describes the application of thermophilic microorganisms for obtaining 6-halogenated purine nucleosides. Biosynthesis of 6-chloropurine-2'-deoxyriboside and 6-chloropurine riboside was achieved by Geobacillus stearothermophilus CECT 43 with a conversion of 90% and 68%, respectively. Furthermore, the selected microorganism was satisfactorily stabilized by immobilization in an agarose matrix. This biocatalyst can be reused at least 70 times without significant loss of activity, obtaining 379mg/L of 6-chloropurine-2'-deoxyriboside. The obtained compounds can be used as antiviral agents.

Complete genome sequence of Bacillus amyloliquefaciens XH7, which exhibits production of purine nucleosides.

Here, we report the complete annotated genome sequence of Bacillus amyloliquefaciens XH7, which is used to produce purine nucleosides in industry. The genome sequence will allow for the characterization of the molecular mechanisms underlying its beneficial properties.

6-oxopurine phosphoribosyltransferase: a target for the development of antimalarial drugs.

Malaria remains the most serious parasitic diseases affecting humans in the world today, resulting in 1-2 million fatalities each year. Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) are the predominant causative agents. Both are responsible for widespread mortality and morbidity and are a serious socio-economic burden, especially for countries in the developing world. One of the most important defences against malaria has been the use of chemotherapeutic drugs (e.g. chloroquine, artemisinins, pyrimethamine) but these have mainly been found by serendipity. Their mechanisms was not understood at the time of their discovery and, even today, are still not unequivocal. For many of these compounds, the parasite is now resistant and, hence, there is an urgent need to develop new therapeutic drugs directed to validated targets. One metabolic pathway crucial for the survival and replication and survival of the parasite is the synthesis of the purine nucleoside monophosphates essential for the production of DNA/RNA molecules. A key enzyme in this pathway is the 6-oxopurine phosphoribosyltransferase (PRTase). The focus of this review is on the identification and characterization of inhibitors of the enzymes from both Pf and Pv as antimalarial drug leads. The acyclic nucleoside phosphonates (ANPs) appear to be excellent candidates because they are good inhibitors of the two Plasmodium enzymes, can be selective compared to the human enzyme, can arrest parasitemia in cell based assays, have low cytotoxicity to the human host cell and, because of their stable carbon-phosphorous bond, are stable within the cell.

Low-molecular-mass purine nucleoside phosphorylase: characterization and application in enzymatic synthesis of nucleoside antiviral drugs.

Purine nucleoside phosphorylase (PNP) that catalyzes the reversible phosphorolysis of various purine nucleosides is widely distributed in prokaryotes and eukaryotes. Four pnp genes from Bacillus subtilis 168, Escherichia coli K-12 and Pseudoalteromonas sp. XM2107 were cloned by PCR and expressed in E. coli XL1-Blue. Recombinant PNPs (rPNPs) were purified by Ni(2+)-NTA chromatography. Compared with other rPNPs, PNP(816) was a low-molecular-mass homotrimer, which exhibited 11-, 4- and 1.5-fold higher values in k (cat)/K (m) using inosine as the substrate at 37°C. The PNP(816) or engineered strain XBlue (pQE-816) had a higher catalytic activity than other rPNPs or engineered strains during the enzymatic synthesis of ribavirin, which suggested that the low-molecular-mass homotrimer derived from microorganisms has higher catalytic activity for synthesis of nucleoside antiviral drugs.

Semisynthesis of 6-chloropurine-2'-deoxyriboside 5'-dimethoxytrityl 3'-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite and its use in the synthesis of fluorescently labeled oligonucleotides.

An efficient enzymatic synthesis of 6-chloropurine-2'-deoxyriboside from the reaction of 6-chloropurine with 2'-deoxycytidine catalyzed by nucleoside-2'-deoxyribosyltransferase (E.C. 2.4.2.6) followed by chemical conversion into the 5'-dimethoxytrityl 3'-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite derivative is described. The phosphoramidite derivative was incorporated site-specifically into an oligonucleotide and used for the introduction of a tethered tetramethylrhodamine-cadaverine conjugate. The availability of an efficient route to 6-chloropurine-2'-deoxyriboside 5'-dimethoxytrityl 3'-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite enables the facile synthesis of oligonucleotides containing a range of functional groups tethered to deoxyadenosine residues.

Enzymatic synthesis of 2'-deoxyadenosine and 6-methylpurine-2'-deoxyriboside by Escherichia coli DH5alpha overexpressing nucleoside phosphorylases from Escherichia coli BL21.

Genes encoding purine nucleoside phosphorylase (deo D), uridine phosphorylase (udp) and thimidine phosphorylase (deo A) from Escherichia coli BL21 were cloned and overexpressed in E. coli DH5alpha. The recombinant strains were employed to synthesize 2'-deoxyadenosine (dAR) and 6-methylpurine-2'-deoxyriboside (MePdR). Experimental parameters such as strains, temperature, pH, reagent concentration and cell mass were optimized. Under the optimal situation, 96% adenine was converted to dAR and 95% 6-methylpurine (MeP) was converted to MePdR in an hour, using 0.2 per thousand (dry wt./v) cell paste as biocatalyst.

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.

An enzymatic transglycosylation of purine bases.

An enzymatic transglycosylation of purine heterocyclic bases employing readily available natural nucleosides or sugar-modified nucleosides as donors of the pentofuranose fragment and recombinant nucleoside phosphorylases as biocatalysts has been investigated. An efficient enzymatic method is suggested for the synthesis of purine nucleosides containing diverse substituents at the C6 and C2 carbon atoms. The glycosylation of N(6)-benzoyladenine and N(2)-acetylguanine and its O(6)-derivatives is not accompanied by deacylation of bases.