Glucosamine-P and rhodococcal ADP-glucose pyrophosphorylases: A hint to (re)discover (actino)bacterial amino sugar metabolism.

Glycogen was described as a temporal storage molecule in rhodococci, interconnecting lipids and carbon availability. The Rhodococcus jostii ADP-glucose pyrophosphorylase (ADP-GlcPPase) kinetic and regulatory properties support this role. Curiously, the enzyme uses glucosamine-1P as alternative substrate. Herein, we report the in-depth study of glucosamine-1P activity and its regulation in two rhodocoocal ADP-GlcPPases, finding that glucosamine-6P (representing a metabolic carbon/nitrogen node) is a critical activator, then reinforcing the role of glycogen as an "intermediary metabolite" in rhodococci. Glucosamine-1P activity in rhodococcal ADP-GlcPPases responds to activation by metabolites improving their catalytic performance, strongly suggesting its metabolic feasibility. This work supports a scenario for new molecules/metabolites discovery and hypothesizes on evolutionary mechanisms underlying enzyme promiscuity opening novel metabolic features in (actino)bacteria.

X-ray crystallographic structure of BshB, the zinc-dependent deacetylase involved in bacillithiol biosynthesis.

Many gram-positive bacteria produce bacillithiol to aid in the maintenance of redox homeostasis and degradation of toxic compounds, including the antibiotic fosfomycin. Bacillithiol is produced via a three-enzyme pathway that includes the action of the zinc-dependent deacetylase BshB. Previous studies identified conserved aspartate and histidine residues within the active site that are involved in metal binding and catalysis, but the enzymatic mechanism is not fully understood. Here we report two X-ray crystallographic structures of BshB from Bacillus subtilis that provide insight into the BshB catalytic mechanism.

A structural and functional analysis of the glycosyltransferase BshA from Staphylococcus aureus: Insights into the reaction mechanism and regulation of bacillithiol production.

Bacillithiol is a glucosamine-derived antioxidant found in several pathogenic Gram-positive bacteria. The compound is involved in maintaining the appropriate redox state within the cell as well as detoxifying foreign agents like the antibiotic fosfomycin. Bacillithiol is produced via the action of three enzymes, including BshA, a retaining GT-B glycosyltransferase that utilizes UDP-N-acetylglucosamine and l-malate to produce N-acetylglucosaminyl-malate. Recent studies suggest that retaining GT-B glycosyltransferases like BshA utilize a substrate-assisted mechanism that goes through an SN i-like transition state. In a previous study, we relied on X-ray crystallography as well as computational simulations to hypothesize the manner in which substrates would bind the enzyme, but several questions about substrate binding and the role of one of the amino acid residues persisted. Another study demonstrated that BshA might be subject to feedback inhibition by bacillithiol, but this phenomenon was not analyzed further to determine the exact mechanism of inhibition. Here we present X-ray crystallographic structures and steady-state kinetics results that help elucidate both of these issues. Our ligand-bound crystal structures demonstrate that the active site provides an appropriate steric and geometric arrangement of ligands to facilitate the substrate-assisted mechanism. Finally, we show that bacillithiol is competitive for UDP-N-acetylglucosamine with a Ki value near 120-130 µM and likely binds within the BshA active site, suggesting that bacillithiol modulates BshA activity via feedback inhibition. The work presented here furthers our understanding of bacillithiol metabolism and can aid in the development of inhibitors to counteract resistance to antibiotics such as fosfomycin.

Mammalian Target of Rapamycin 2 (MTOR2) and C-MYC Modulate Glucosamine-6-Phosphate Synthesis in Glioblastoma (GBM) Cells Through Glutamine: Fructose-6-Phosphate Aminotransferase 1 (GFAT1).

Glucose and glutamine are two essential ingredients for cell growth. Glycolysis and glutaminolysis can be linked by glutamine: fructose-6-phosphate aminotransferase (GFAT, composed of GFAT1 and GFAT2) that catalyzes the synthesis of glucosamine-6-phosphate and glutamate by using fructose-6-phosphate and glutamine as substrates. The role of mammalian target of rapamycin (MTOR, composed of MTOR1 and MTOR2) in regulating glycolysis has been explored in human cancer cells. However, whether MTOR can interact with GFAT to regulate glucosamine-6-phosphate is poorly understood. In this study, we report that GFAT1 is essential to maintain the malignant features of GBM cells. And MTOR2 rather than MTOR1 plays a robust role in promoting GFAT1 protein activity, and accelerating the progression of glucosamine-6-phosphate synthesis, which is not controlled by the PI3K/AKT signaling. Intriguingly, high level of glucose or glutamine supply promotes MTOR2 protein activity. In turn, up-regulating glycolytic and glutaminolytic metabolisms block MTOR dimerization, enhancing the release of MTOR2 from the MTOR complex. As a transcriptional factor, C-MYC, directly targeted by MTOR2, promotes the relative mRNA expression level of GFAT1. Notably, our data reveal that GFAT1 immunoreactivity is positively correlated with the malignant grades of glioma patients. Kaplan-Meier assay reveals the correlations between patients' 5-year survival and high GFAT1 protein expression. Taken together, we propose that the MTOR2/C-MYC/GFAT1 axis is responsible for the modulation on the crosstalk between glycolysis and glutaminolysis in GBM cells. Under the condition of accelerated glycolytic and/or glutaminolytic metabolisms, the MTOR2/C-MYC/GFAT1 axis will be up-regulated in GBM cells.

Redox regulation by reversible protein S-thiolation in Gram-positive bacteria.

Low molecular weight (LMW) thiols play an important role as thiol-cofactors for many enzymes and are crucial to maintain the reduced state of the cytoplasm. Most Gram-negative bacteria utilize glutathione (GSH) as major LMW thiol. However, in Gram-positive Actinomycetes and Firmicutes alternative LMW thiols, such as mycothiol (MSH) and bacillithiol (BSH) play related roles as GSH surrogates, respectively. Under conditions of hypochlorite stress, MSH and BSH are known to form mixed disulfides with protein thiols, termed as S-mycothiolation or S-bacillithiolation that function in thiol-protection and redox regulation. Protein S-thiolations are widespread redox-modifications discovered in different Gram-positive bacteria, such as Bacillus and Staphylococcus species, Mycobacterium smegmatis, Corynebacterium glutamicum and Corynebacterium diphtheriae. S-thiolated proteins are mainly involved in cellular metabolism, protein translation, redox regulation and antioxidant functions with some conserved targets across bacteria. The reduction of protein S-mycothiolations and S-bacillithiolations requires glutaredoxin-related mycoredoxin and bacilliredoxin pathways to regenerate protein functions. In this review, we present an overview of the functions of mycothiol and bacillithiol and their physiological roles in protein S-bacillithiolations and S-mycothiolations in Gram-positive bacteria. Significant progress has been made to characterize the role of protein S-thiolation in redox-regulation and thiol protection of main metabolic and antioxidant enzymes. However, the physiological roles of the pathways for regeneration are only beginning to emerge as well as their interactions with other cellular redox systems. Future studies should be also directed to explore the roles of protein S-thiolations and their redox pathways in pathogenic bacteria under infection conditions to discover new drug targets and treatment options against multiple antibiotic resistant bacteria.

The aldehyde dehydrogenase AldA contributes to the hypochlorite defense and is redox-controlled by protein S-bacillithiolation in Staphylococcus aureus.

Staphylococcus aureus produces bacillithiol (BSH) as major low molecular weight (LMW) thiol which functions in thiol-protection and redox-regulation by protein S-bacillithiolation under hypochlorite stress. The aldehyde dehydrogenase AldA was identified as S-bacillithiolated at its active site Cys279 under NaOCl stress in S. aureus. Here, we have studied the expression, function, redox regulation and structural changes of AldA of S. aureus. Transcription of aldA was previously shown to be regulated by the alternative sigma factor SigmaB. Northern blot analysis revealed SigmaB-independent induction of aldA transcription under formaldehyde, methylglyoxal, diamide and NaOCl stress. Deletion of aldA resulted in a NaOCl-sensitive phenotype in survival assays, suggesting an important role of AldA in the NaOCl stress defense. Purified AldA showed broad substrate specificity for oxidation of several aldehydes, including formaldehyde, methylglyoxal, acetaldehyde and glycol aldehyde. Thus, AldA could be involved in detoxification of aldehyde substrates that are elevated under NaOCl stress. Kinetic activity assays revealed that AldA is irreversibly inhibited under H2O2 treatment in vitro due to overoxidation of Cys279 in the absence of BSH. Pre-treatment of AldA with BSH prior to H2O2 exposure resulted in reversible AldA inactivation due to S-bacillithiolation as revealed by activity assays and BSH-specific Western blot analysis. Using molecular docking and molecular dynamic simulation, we further show that BSH occupies two different positions in the AldA active site depending on the AldA activation state. In conclusion, we show here that AldA is an important target for S-bacillithiolation in S. aureus that is up-regulated under NaOCl stress and functions in protection under hypochlorite stress.

Critical Minireview: The Fate of tRNACys during Oxidative Stress in Bacillus subtilis.

Oxidative stress occurs when cells are exposed to elevated levels of reactive oxygen species that can damage biological molecules. One bacterial response to oxidative stress involves disulfide bond formation either between protein thiols or between protein thiols and low-molecular-weight (LMW) thiols. Bacillithiol was recently identified as a major low-molecular-weight thiol in Bacillus subtilis and related Firmicutes. Four genes (bshA, bshB1, bshB2, and bshC) are involved in bacillithiol biosynthesis. The bshA and bshB1 genes are part of a seven-gene operon (ypjD), which includes the essential gene cca, encoding CCA-tRNA nucleotidyltransferase. The inclusion of cca in the operon containing bacillithiol biosynthetic genes suggests that the integrity of the 3' terminus of tRNAs may also be important in oxidative stress. The addition of the 3' terminal CCA sequence by CCA-tRNA nucleotidyltransferase to give rise to a mature tRNA and functional molecules ready for aminoacylation plays an essential role during translation and expression of the genetic code. Any defects in these processes, such as the accumulation of shorter and defective tRNAs under oxidative stress, might exert a deleterious effect on cells. This review summarizes the physiological link between tRNACys regulation and oxidative stress in Bacillus.

Core pathways operating during methylotrophy of Bacillus methanolicus MGA3 and induction of a bacillithiol-dependent detoxification pathway upon formaldehyde stress.

Bacillus methanolicus MGA3 is a model facultative methylotroph of interest for fundamental research and biotechnological applications. Previous research uncovered a number of pathways potentially involved in one-carbon substrate utilization. Here, we applied dynamic (13) C labeling to elucidate which of these pathways operate during growth on methanol and to uncover potentially new ones. B. methanolicus MGA3 uses the assimilatory and dissimilatory ribulose monophosphate (RuMP) cycles for conversion of the central but toxic intermediate formaldehyde. Additionally, the operation of two cofactor-dependent formaldehyde oxidation pathways with distinct roles was revealed. One is dependent on tri- and tetraglutamylated tetrahydrofolate (THF) and is involved in formaldehyde oxidation during growth on methanol. A second pathway was discovered that is dependent on bacillithiol, a thiol cofactor present also in other Bacilli where it is known to function in redox-homeostasis. We show that bacillithiol-dependent formaldehyde oxidation is activated upon an upshift in formaldehyde induced by a substrate switch from mannitol to methanol. The genes and the corresponding enzymes involved in the biosynthesis of bacillithiol were identified by heterologous production of bacillithiol in Escherichia coli. The presented results indicate metabolic plasticity of the methylotroph allowing acclimation to fluctuating intracellular formaldehyde concentrations.

Bacillithiol has a role in Fe-S cluster biogenesis in Staphylococcus aureus.

Staphylococcus aureus does not produce the low-molecular-weight (LMW) thiol glutathione, but it does produce the LMW thiol bacillithiol (BSH). To better understand the roles that BSH plays in staphylococcal metabolism, we constructed and examined strains lacking BSH. Phenotypic analysis found that the BSH-deficient strains cultured either aerobically or anaerobically had growth defects that were alleviated by the addition of exogenous iron (Fe) or the amino acids leucine and isoleucine. The activities of the iron-sulfur (Fe-S) cluster-dependent enzymes LeuCD and IlvD, which are required for the biosynthesis of leucine and isoleucine, were decreased in strains lacking BSH. The BSH-deficient cells also had decreased aconitase and glutamate synthase activities, suggesting a general defect in Fe-S cluster biogenesis. The phenotypes of the BSH-deficient strains were exacerbated in strains lacking the Fe-S cluster carrier Nfu and partially suppressed by multicopy expression of either sufA or nfu, suggesting functional overlap between BSH and Fe-S carrier proteins. Biochemical analysis found that SufA bound and transferred Fe-S clusters to apo-aconitase, verifying that it serves as an Fe-S cluster carrier. The results presented are consistent with the hypothesis that BSH has roles in Fe homeostasis and the carriage of Fe-S clusters to apo-proteins in S. aureus.

Regulation of Bacillus subtilis bacillithiol biosynthesis operons by Spx.

Bacillithiol is the major low molecular mass thiol produced by many firmicutes bacteria, including the model organism Bacillus subtilis and pathogens such as Bacillus anthracis and Staphylococcus aureus. We have previously shown that four genes (bshA, bshB1, bshB2 and bshC) are involved in bacillithiol biosynthesis. Here, we report that these four genes are encoded within three, unlinked operons all expressed from canonical σ(A)-dependent promoters as determined by 5'RACE (rapid amplification of cDNA ends). The bshA and bshB1 genes are embedded within a seven-gene operon additionally including mgsA, encoding methylglyoxal synthase, and the essential genes cca and birA, encoding tRNA nucleotidyltransferase (CCA transferase) and biotin-protein ligase, respectively. The bshB2 gene is co-transcribed with unknown function genes, while bshC is expressed both as part of a two-gene operon (with the upstream putative pantothenate biosynthesis gene ylbQ) and from its own promoter. All three operons are expressed at a reduced level in an spx null mutant, consistent with a direct role of Spx as a transcriptional activator for these operons, and all three operons are induced by the thiol oxidant diamide. In contrast with other Spx-regulated genes characterized to date, the effects of Spx on basal expression and diamide-stimulated expression appear to be independent of Cys10 in the redox centre of Spx. Consistent with the role of Spx as an activator of bacillithiol biosynthetic genes, cellular levels of bacillithiol are reduced several-fold in an spx null mutant.

Distribution and infection-related functions of bacillithiol in Staphylococcus aureus.

Bacillithiol (Cys-GlcN-malate, BSH) serves as a major low molecular weight thiol in low GC Gram-positive bacteria including Bacillus species and a variety of Staphylococcus aureus strains. These bacteria do not produce glutathione (GSH). In this study, HPLC analyses were used to determine BSH levels in different S. aureus strains. Furthermore, the role of BSH in the resistance against oxidants and antibiotics and its function in virulence was investigated. We and others (Newton, G.L., Fahey, R.C., Rawat, M., 2012. Microbiology 158, 1117-1126) found that BSH is not produced by members of the S. aureus NCTC8325 lineage, such as strains 8325-4 and SH1000. Using bioinformatics we show that the BSH-biosynthetic gene bshC is disrupted by an 8-bp duplication in S. aureus NCTC8325. The functional bshC-gene from BSH-producing S. aureus Newman (NWMN_1087) was expressed in S. aureus 8325-4 to reconstitute BSH-synthesis. Comparison of the BSH-producing and BSH-minus strains revealed higher resistance of the BSH-producing strain against the antibiotic fosfomycin and the oxidant hypochlorite but not against hydrogen peroxide or diamide. In addition, a higher bacterial load of the BSH-producing strain was detected in human upper-airway epithelial cells and murine macrophages. This indicates a potential role of BSH in protection of S. aureus during infection.

A new bifunctional chitosanase enzyme from Streptomyces sp. and its application in production of antioxidant chitooligosaccharides.

Chitosanases produced by microbes and plants are getting attention to explore vastly available marine waste. Chitooligosaccharides and glucosamine can be produced using chitosanase enzyme and have applications in food, pharma and other industries. A potential microbial chitosanase source was found after isolation and screening of chitosan degrading microbes from garden soil. An isolate, designated as C6 produced chitosanase enzyme upon induction by chitosan substrates. Production of 6 U/ml of chitosanase enzyme was achieved from this isolate on chitosan minimal salt broth medium at 32 °C after 3 days of growth. The enzyme was able to hydrolyse both chitosan and cellulosic substrates. Enzymatic production of D -glucosamine and chitooligosaccharides were studied with various chitosan substrates using crude enzyme. The yield of glucosamine was found to be 40% after 2 h of reaction at 40 °C, and chitosan oligomers were produced having two to six polymerizations at 60 °C reaction temperature. The hydrolysates showed 50% antioxidant activity as compared to ascorbic acid.

Characterization of the N-acetyl-α-D-glucosaminyl l-malate synthase and deacetylase functions for bacillithiol biosynthesis in Bacillus anthracis .

Bacillithiol (Cys-GlcN-malate, BSH) has recently been identified as a novel low-molecular weight thiol in Bacillus anthracis, Staphylococcus aureus, and several other Gram-positive bacteria lacking glutathione and mycothiol. We have now characterized the first two enzymes for the BSH biosynthetic pathway in B. anthracis, which combine to produce α-d-glucosaminyl l-malate (GlcN-malate) from UDP-GlcNAc and l-malate. The structure of the GlcNAc-malate intermediate has been determined, as have the kinetic parameters for the BaBshA glycosyltransferase (→GlcNAc-malate) and the BaBshB deacetylase (→GlcN-malate). BSH is one of only two natural products reported to contain a malyl glycoside, and the crystal structure of the BaBshA-UDP-malate ternary complex, determined in this work at 3.3 Å resolution, identifies several active-site interactions important for the specific recognition of l-malate, but not other α-hydroxy acids, as the acceptor substrate. In sharp contrast to the structures reported for the GlcNAc-1-d-myo-inositol-3-phosphate synthase (MshA) apo and ternary complex forms, there is no major conformational change observed in the structures of the corresponding BaBshA forms. A mutant strain of B. anthracis deficient in the BshA glycosyltransferase fails to produce BSH, as predicted. This B. anthracis bshA locus (BA1558) has been identified in a transposon-site hybridization study as required for growth, sporulation, or germination [Day, W. A., Jr., Rasmussen, S. L., Carpenter, B. M., Peterson, S. N., and Friedlander, A. M. (2007) J. Bacteriol. 189, 3296-3301], suggesting that the biosynthesis of BSH could represent a target for the development of novel antimicrobials with broad-spectrum activity against Gram-positive pathogens like B. anthracis. The metabolites that function in thiol redox buffering and homeostasis in Bacillus are not well understood, and we present a composite picture based on this and other recent work.

Biosynthesis and functions of bacillithiol, a major low-molecular-weight thiol in Bacilli.

Bacillithiol (BSH), the alpha-anomeric glycoside of L-cysteinyl-D-glucosamine with L-malic acid, is a major low-molecular-weight thiol in Bacillus subtilis and related bacteria. Here, we identify genes required for BSH biosynthesis and provide evidence that the synthetic pathway has similarities to that established for the related thiol (mycothiol) in the Actinobacteria. Consistent with a key role for BSH in detoxification of electrophiles, the BshA glycosyltransferase and BshB1 deacetylase are encoded in an operon with methylglyoxal synthase. BshB1 is partially redundant in function with BshB2, a deacetylase of the LmbE family. Phylogenomic profiling identified a conserved unknown function protein (COG4365) as a candidate cysteine-adding enzyme (BshC) that co-occurs in genomes also encoding BshA, BshB1, and BshB2. Additional evolutionarily linked proteins include a thioredoxin reductase homolog and two thiol:disulfide oxidoreductases of the DUF1094 (CxC motif) family. Mutants lacking BshA, BshC, or both BshB1 and BshB2 are devoid of BSH. BSH is at least partially redundant in function with other low-molecular-weight thiols: redox proteomics indicates that protein thiols are largely reduced even in the absence of BSH. At the transcriptional level, the induction of genes controlled by two thiol-based regulators (OhrR, Spx) occurs normally. However, BSH null cells are significantly altered in acid and salt resistance, sporulation, and resistance to electrophiles and thiol reactive compounds. Moreover, cells lacking BSH are highly sensitive to fosfomycin, an epoxide-containing antibiotic detoxified by FosB, a prototype for bacillithiol-S-transferase enzymes.

Genetic analysis of genes involved in synthesis of modified 4-amino-4,6-dideoxyglucose in flagellin of Pseudomonas syringae pv. tabaci.

Glycosylation of flagellin contributes to swimming and swarming motilities, adhesion ability, and consequently virulence in Pseudomonas syringae pv. tabaci 6605. Glycans attached to six serine residues are located in the central region of the flagellin polypeptide. The glycan structure at position Ser 201 was recently revealed to consist of two L-rhamnoses and one modified 4-amino-4,6-dideoxyglucose (viosamine). To clarify the mechanisms for glycosylation of modified viosamine, genes encoding dTDP-viosamine aminotransferase (vioA), dTDP-viosamine acetyltransferase (vioB), and viosamine-derivative transferase (vioT) were isolated and defective mutants were generated. MALDI-TOF-MS analysis of a lysyl endopeptidase-digested peptide including all six glycosylation sites from each flagellin indicated that the molecular masses of the three flagellin mutants were reduced with highly heterogeneous patterns at regular intervals of 146 Da in the mass range from m/z 13,819 to 15,732. The data indicated that the glycopeptides obtained from mutants had glycans consisting only of deoxyhexose instead of the flagellin glycans including the viosamine derivatives determined previously. The motility and virulence on host tobacco leaves were strongly impaired in the Delta vioA mutant and were weakly reduced in the Delta vioB and Delta vioT mutant strains. These results suggest that the genes vioA, vioB, and vioT are essential for glycosylation of flagellin, and accordingly are required for bacterial virulence.

Characterization of the complete zwittermicin A biosynthesis gene cluster from Bacillus cereus.

Bacillus cereus UW85 produces the linear aminopolyol antibiotic zwittermicin A (ZmA). This antibiotic has diverse biological activities, such as suppression of disease in plants caused by protists, inhibition of fungal and bacterial growth, and amplification of the insecticidal activity of the toxin protein from Bacillus thuringiensis. ZmA has an unusual chemical structure that includes a d amino acid and ethanolamine and glycolyl moieties, as well as having an unusual terminal amide that is generated from the modification of the nonproteinogenic amino acid beta-ureidoalanine. The diverse biological activities and unusual structure of ZmA have stimulated our efforts to understand how this antibiotic is biosynthesized. Here, we present the identification of the complete ZmA biosynthesis gene cluster from B. cereus UW85. A nearly identical gene cluster is identified on a plasmid from B. cereus AH1134, and we show that this strain is also capable of producing ZmA. Bioinformatics and biochemical analyses of the ZmA biosynthesis enzymes strongly suggest that ZmA is initially biosynthesized as part of a larger metabolite that is processed twice, resulting in the formation of ZmA and two additional metabolites. Additionally, we propose that the biosynthesis gene cluster for the production of the amino sugar kanosamine is contained within the ZmA biosynthesis gene cluster in B. cereus UW85.

Expression of glucosamine trisaccharides on the rat uterine surface is altered by clomiphene citrate. III. Relationship with implantation regimes and pregnancy.

The present study further elucidates the complex effects of a commonly-prescribed fertility drug upon a target organ in an animal model. In the human condition, its effects are rarely observed without the influence of endogenous ovarian hormones. The aim of the study was to investigate how the administration of a single dose of clomiphene citrate (CC) given prior to an implantation-priming sequence of ovarian hormones would affect the expression of surface oligosaccharides and membrane architecture of uterine epithelium. Ovariectomized rats were given a single dose of either 0.25 mg or 1.25 mg of CC prior to a hormone-priming regime of progesterone (P4) for 3 days with a single additional administration of oestradiol 17beta (E2) on day 3. Animals were killed 24 h after final treatment. Uterine tissue was labelled with the lectin Phytolacca americana conjugated with avidin, subsequently labelled with biotinylated ferritin and prepared for transmission electron microscopy. Results indicate that CC, when administered prior to the implantation hormone-priming regime, is able to act as a super oestrogen and upregulates expression of oligosaccharides on the plasma membrane surface and increases the density and length of microvilli on the surface of the cells when compared with other treatment regimes. Understanding of the effects of CC at the uterine level at the time of implantation enables manipulation of uterine receptivity to control fertility and to improve the outcome of assisted reproductive procedures.

Expression, purification, and characterization of two N,N-dimethyltransferases, tylM1 and desVI, involved in the biosynthesis of mycaminose and desosamine.

Methylation catalyzed by an S-adenosylmethionine- (AdoMet-) dependent methyltransferase is an effective means to alter the hydrophilicity and/or nucleophilicity of a molecule. While a large number of enzymes capable of catalyzing methylation at carbon, oxygen, sulfur, and nitrogen atoms are known, only a few are able to catalyze N,N-dimethylation. Mycaminose and desosamine are aminohexoses found in several macrolide antibiotics, such as tylosin and methymycin, respectively. Both sugars contain a C-3 N,N-dimethylamino group which has been shown to confer the biological activity of these unusual sugars. Recently, sequence analysis as well as genetic studies has led to the assignment of tylM1 in the tylosin biosynthetic gene cluster and desVI in the methymycin biosynthetic gene cluster as genes encoding the corresponding N,N-dimethyltransferases. To verify the proposed roles of the tylM1 and desVI genes, we have overexpressed and purified their encoded products, synthesized the predicted substrates, and characterized the catalytic function of these proteins. Our studies showed that TylM1 and DesVI are homodimeric proteins and have nearly identical biochemical properties. These enzymes do not have strong preference for binding either the unmethylated substrate or the monomethylated intermediate. It is the chemical reactivity of the nitrogen functional group that determines the relative rate of a particular methylation step. Thus, our results not only establish TylM1 and DesVI as new members of a small family of enzymes that are capable of catalyzing N,N-dimethylation of an amino group but also provide evidence indicating that the methylation catalyzed by AdoMet-dependent methyltransferases proceeds in a stepwise manner and is nucleophilic in nature.

Regulation of glutamine:fructose-6-phosphate amidotransferase activity by high glucose and transforming growth factor beta in rat mesangial cells.

BACKGROUND: The hexosamine biosynthesis pathway acts as a cellular glucose sensor and mediates many of the adverse effects of glucose. Increased flux through this pathway results in insulin resistance in rat fibroblasts and transgenic mice and upregulation of transforming growth factor beta (TGF-beta) transcriptional activity in rat kidney cells. The first and rate-limiting step in this pathway, which is responsible for the metabolism of glucose to glucosamine, is catalyzed by glutamine:fructose-6-phosphate amidotransferase (GFA). METHODS: Because of the known effects of hyperglycemia on mesangial cell (MC) function and growth factor regulation, we examined the regulation of GFA by glucose and TGF-beta in cultured SV40 rat MCs. GFA activity was assayed in cytosolic extracts of MCs using high-performance liquid chromatography. RESULTS: Culturing in 10 and 25 mM of glucose for 24 hours resulted in 33.4% (P < 0.025) and 43.5% (P < 0.05) decreases in GFA activity when compared with cells cultured at 1 to 5 mM of glucose. The downregulation in GFA activity by high glucose (HG) required at least 6 hours in culture and persisted for several days. HG effects were not a result of osmolar changes or glucose-induced differences in glucose uptake. Like HG, treatment of MCs with TGF-beta (2 ng/mL) for 4 hours resulted in a 30% (P < 0.05) decrease in GFA activity in cells cultured at 1 mM glucose, but the effects of TGF-beta were not additive to those of HG. TGF-beta-mediated downregulation of GFA activity was inhibited by a TGF-beta-neutralizing antibody, but HG's effects were not. Insulin-like growth factor-1 (IGF-1) had similar effects as TGF-beta, but GFA activity was not regulated by angiotensin II. CONCLUSIONS: GFA activity is downregulated by HG, TGF-beta, and IGF-1 in rat MCs. Downregulation of this cellular glucose sensor may be a protective mechanism against the harmful effects of excess glucose as seen in diabetes.

Reduced O glycosylation of Sp1 is associated with increased proteasome susceptibility.

Sp1 is a ubiquitously expressed transcription factor that is particularly important for the regulation of TATA-less genes that encode housekeeping proteins. Most growth factors and receptors are also encoded by such genes. Sp1 is multiply O glycosylated by covalent linkage of the monosaccharide N-acetylglucosamine (O-GlcNAc) to serine and threonine residues. Based on an earlier observation that growth factor gene transcription can be regulated by glucose and glucosamine in vascular smooth muscle cells, we determined whether Sp1 glycosylation could be regulated and if this modification altered Sp1 function. We found that Sp1 becomes hyperglycosylated when cells are exposed to 5 mM glucosamine, whereas under glucose starvation, stimulation with cyclic AMP (cAMP) results in nearly complete deglycosylation of this protein. Correlating with this hypoglycosylated state, Sp1 is rapidly proteolytically degraded by an enzyme(s) that can be inhibited by specific proteasome inhibitors, lactacystin and LLnL. Treatment of cells with glucose or glucosamine protects Sp1 from cAMP-mediated degradation, whereas blockade of glucosamine synthesis abrogates glucose but not glucosamine protection. This effect on Sp1 is specific, in that the Stat-3 and E2F transcription factors did not undergo degradation under these conditions. The O-GlcNAc modification of Sp1 may play a role as a nutritional checkpoint. In the absence of adequate nutrition, Sp1 becomes hypoglycosylated and thereby subject to proteasome degradation. This process could potentially result in reduced general transcription, thereby conserving nutrients.