Biochemical and Biophysical Characterization of Recombinant Human 3-Phosphoglycerate Dehydrogenase.

The human enzyme D-3-phosphoglycerate dehydrogenase (hPHGDH) catalyzes the reversible dehydrogenation of 3-phosphoglycerate (3PG) into 3-phosphohydroxypyruvate (PHP) using the NAD/NADH redox cofactor, the first step in the phosphorylated pathway producing L-serine. We focused on the full-length enzyme that was produced in fairly large amounts in cells; the effect of pH, temperature and ligands on hPHGDH activity was studied. The forward reaction was investigated on 3PG and alternative carboxylic acids by employing two coupled assays, both removing the product PHP; 3PG was by far the best substrate in the forward direction.

Description of the bacterial RNA polymerase inhibitor GE23077-producer sp. NRRL B-65521 as sp. nov.

The filamentous actinomycete that produces the antibiotic GE23077 was isolated by the Lepetit Research Group from a soil sample collected in Thailand, and it was classified as a member of the genus on the basis of its morphology and cell-wall composition. Phylogenetic analysis based on 16S rRNA gene sequences indicated that this strain formed a distinct monophyletic line within the genus and it was most closely related to DSM 45347 (99.31 % similarity) and DSM 44485 (98.

L-serine synthesis via the phosphorylated pathway in humans.

L-serine is a nonessential amino acid in eukaryotic cells, used for protein synthesis and in producing phosphoglycerides, glycerides, sphingolipids, phosphatidylserine, and methylenetetrahydrofolate. Moreover, L-serine is the precursor of two relevant coagonists of NMDA receptors: glycine (through the enzyme serine hydroxymethyltransferase), which preferentially acts on extrasynaptic receptors and D-serine (through the enzyme serine racemase), dominant at synaptic receptors. The cytosolic "phosphorylated pathway" regulates de novo biosynthesis of L-serine, employing 3-phosphoglycerate generated by glycolysis and the enzymes 3-phosphoglycerate dehydrogenase, phosphoserine aminotransferase, and phosphoserine phosphatase (the latter representing the irreversible step).

New Molecular Tools for Regulation and Improvement of A40926 Glycopeptide Antibiotic Production in ATCC 39727.

Genome sequencing has revealed that spp. represent a still largely unexplored source of specialized metabolites. ATCC 39727 is the most studied representative species since it produces the glycopeptide antibiotic (GPA) A40926 - the precursor of the clinically relevant antibiotic dalbavancin, approved by the FDA in 2014 for the treatment of acute skin infections caused by multi-drug resistant Gram-positive pathogens.

D-amino acids in foods.

With the only exception of glycine, all amino acids exist in two specular structures which are mirror images of each other, called D-(dextro) and L-(levo) enantiomers. During evolution, L-amino acids were preferred for protein synthesis and main metabolism; however, the D-amino acids (D-AAs) acquired different and specific functions in different organisms (from playing a structural role in the peptidoglycan of the bacterial cell wall to modulating neurotransmission in mammalian brain). With the advent of sophisticated and sensitive analytical techniques, it was established during the past few decades that many foods contain considerable amounts of D-AAs: we consume more than 100 mg of D-AAs every day.

Magnetic Nanoconjugated Teicoplanin: A Novel Tool for Bacterial Infection Site Targeting.

Nanoconjugated antibiotics can be regarded as next-generation drugs as they possess remarkable potential to overcome multidrug resistance in pathogenic bacteria. Iron oxide nanoparticles (IONPs) have been extensively used in the biomedical field because of their biocompatibility and magnetic properties. More recently, IONPs have been investigated as potential nanocarriers for antibiotics to be magnetically directed to/recovered from infection sites.

Specificity of Induction of Glycopeptide Antibiotic Resistance in the Producing Actinomycetes.

Glycopeptide antibiotics are drugs of last resort for treating severe infections caused by Gram-positive pathogens. It is widely believed that glycopeptide-resistance determinants ( genes) are ultimately derived from the producing actinomycetes. We hereby investigated the relationship between the antimicrobial activity of vancomycin and teicoplanins and their differential ability to induce gene expression in —the producer of teicoplanin—and —the producer of the teicoplanin-like A40926.

Old and new glycopeptide antibiotics: From product to gene and back in the post-genomic era.

Glycopeptide antibiotics are drugs of last resort for treating severe infections caused by multi-drug resistant Gram-positive pathogens. First-generation glycopeptides (vancomycin and teicoplanin) are produced by soil-dwelling actinomycetes. Second-generation glycopeptides (dalbavancin, oritavancin, and telavancin) are semi-synthetic derivatives of the progenitor natural products.

Metagenomics: novel enzymes from non-culturable microbes.

In the transition to the post-petroleum economy, there is a growing demand for novel enzymes with high process performances to replace traditional chemistry with a more 'green' approach. To date, microorganisms encompass the richest source of industrial biocatalysts, but the Earth-living microbiota remains largely untapped by using traditional isolation and cultivation methods. Metagenomics, which is culture independent, represents a powerful tool for discovering novel enzymes from unculturable microorganisms.

Classification of Actinoplanes sp. ATCC 33076, an actinomycete that produces the glycolipodepsipeptide antibiotic ramoplanin, as Actinoplanes ramoplaninifer sp. nov.

Strain ATCC 33076, which produces the antibiotic ramoplanin, was isolated from a soil sample collected in India, and it was classified as a member of the genus Actinoplanes on the basis of morphology and cell-wall composition. A phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain forms a distinct clade within the genus Actinoplanes, and it is most closely related to Actinoplanes deccanensis IFO 13994 (98.71 % similarity) and Actinoplanes atraurantiacus Y16 (98.

Extraction and Analysis of Peptidoglycan Cell Wall Precursors.

Extraction and analysis by LC-MS of peptidoglycan precursors represent a valuable method to study antibiotic mode of action and resistance in bacteria. Here, we describe how to apply this method for: (1) testing the action of different classes of antibiotics inhibiting cell wall biosynthesis in Bacillus megaterium; (2) studying the mechanism of self-resistance in mycelial actinomycetes producing glycopeptide antibiotics.

Classification of Nonomuraea sp. ATCC 39727, an actinomycete that produces the glycopeptide antibiotic A40926, as Nonomuraea gerenzanensis sp. nov.

Strain ATCC 39727, which produces the antibiotic A40926 (the natural precursor of the antibiotic dalbavancin), was isolated from a soil sample collected in India, and it was originally classified as a member of the genus Actinomadura on the base of morphology and cell-wall composition. A phylogenetic analysis based on 16S rRNA gene sequences indicates that the strain forms a distinct clade within the genus Nonomuraea, and it is most closely related to Nonomuraea angiospora DSM 43173T (98.72 % similarity) and Nonomuraea jabiensis A4036T (98.

Antibacterial Discovery and Development: From Gene to Product and Back.

Concern over the reports of antibiotic-resistant bacterial infections in hospitals and in the community has been publicized in the media, accompanied by comments on the risk that we may soon run out of antibiotics as a way to control infectious disease. Infections caused by Enterococcus faecium, Staphylococcus aureus, Klebsiella species, Clostridium difficile, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, and other Enterobacteriaceae species represent a major public health burden. Despite the pharmaceutical sector's lack of interest in the topic in the last decade, microbial natural products continue to represent one of the most interesting sources for discovering and developing novel antibacterials.

Relationship between glycopeptide production and resistance in the actinomycete Nonomuraea sp. ATCC 39727.

Glycopeptides and β-lactams inhibit bacterial peptidoglycan synthesis in Gram-positive bacteria; resistance to these antibiotics is studied intensively in enterococci and staphylococci because of their relevance to infectious disease. Much less is known about antibiotic resistance in glycopeptide-producing actinomycetes that are likely to represent the evolutionary source of resistance determinants found in bacterial pathogens. Nonomuraea sp.

Old and New Glycopeptide Antibiotics: Action and Resistance.

Glycopeptides are considered antibiotics of last resort for the treatment of life-threatening infections caused by relevant Gram-positive human pathogens, such as Staphylococcus aureus, Enterococcus spp. and Clostridium difficile. The emergence of glycopeptide-resistant clinical isolates, first among enterococci and then in staphylococci, has prompted research for second generation glycopeptides and a flurry of activity aimed at understanding resistance mechanisms and their evolution.

Streptomyces spp. as efficient expression system for a D,D-peptidase/D,D-carboxypeptidase involved in glycopeptide antibiotic resistance.

Background: VanYn, encoded by the dbv7 gene (also known as vanYn) of the biosynthetic cluster devoted to A40926 production, is a novel protein involved in the mechanism of self-resistance in Nonomuraea sp. ATCC 39727. This filamentous actinomycete is an uncommon microorganism, difficult-to-handle but biotechnologically valuable since it produces the glycopeptide antibiotic A40926, which is the precursor of the second-generation dalbavancin in phase III of clinical development.

Actinoplanes teichomyceticus ATCC 31121 as a cell factory for producing teicoplanin.

Background: Teicoplanin is a glycopeptide antibiotic used clinically in Europe and in Japan for the treatment of multi-resistant Gram-positive infections. It is produced by fermenting Actinoplanes teichomyceticus. The pharmaceutically active principle is teicoplanin A2, a complex of compounds designated T-A2-1-A2-5 differing in the length and branching of the fatty acid moiety linked to the glucosamine residue on the heptapeptide scaffold.

β-Lactam and glycopeptide antibiotics: first and last line of defense?

Most infections are caused by bacteria, many of which are ever-evolving and resistant to nearly all available antibiotics. β-Lactams and glycopeptides are used to combat these infections by inhibiting bacterial cell-wall synthesis. This mechanism remains an interesting target in the search for new antibiotics in light of failed genomic approaches and the limited input of major pharmaceutical companies.

Methods for the genetic manipulation of Nonomuraea sp. ATCC 39727.

Nonomuraea sp. ATCC 39727 belongs to the Streptosporangiaceae family of filamentous actinomycetes. This microorganism produces the teicoplanin-like glycopeptide A40926, which is the starting material for the synthesis of the second-generation glycopeptide dalbavancin.

Novel mechanism of glycopeptide resistance in the A40926 producer Nonomuraea sp. ATCC 39727.

In glycopeptide-resistant enterococci and staphylococci, high-level resistance is achieved by replacing the C-terminal d-alanyl-d-alanine of lipid II with d-alanyl-d-lactate, thus reducing glycopeptide affinity for cell wall targets. Reorganization of the cell wall in these organisms is directed by the vanHAX gene cluster. Similar self-resistance mechanisms have been reported for glycopeptide-producing actinomycetes.

Protoplast preparation and reversion to the normal filamentous growth in antibiotic-producing uncommon actinomycetes.

Protoplast preparation, regeneration and fusion represent essential tools for those poorly studied biotechnologically valuable microorganisms inapplicable with the current molecular biology protocols. The protoplast production and regeneration method developed for Planobispora rosea and using the combination of hen egg-white lysozyme (HEWL) and Streptomyces globisporus mutanolysin was applied to a set of antibiotic-producing filamentous actinomycetes belonging to the Streptosporangiaceae, Micromonosporaceae and Streptomycetaceae. 10(7)-10(9) protoplasts were obtained from 100 ml of culture, after incubation times in the digestion solution ranging from a few hours to 1 or 2 days depending on the strain.

Glycine oxidase from Bacillus subtilis: role of histidine 244 and methionine 261.

The reactions of several mutants at position 244 and 261 of bacterial glycine oxidase (GO) were studied by stopped-flow and steady-state kinetic methods. Substituting H244 with phenylalanine, glutamate, and glutamine and M261 with histidine and tyrosine did not affect the expression of GO and the physicochemical properties of bound FAD. All the H244 and M261 mutants of GO we prepared retained activity in both steady-state and stopped-flow kinetic studies, indicating they do not serve as key elements in glycine and sarcosine oxidation.

Evolution of an acylase active on cephalosporin C.

Semisynthetic cephalosporins are synthesized from 7-amino cephalosporanic acid, which is produced by chemical deacylation or by a two-step enzymatic process of the natural antibiotic cephalosporin C. The known acylases take glutaryl-7-amino cephalosporanic acid as a primary substrate, and their specificity and activity are too low for cephalosporin C. Starting from a known glutaryl-7-amino cephalosporanic acid acylase as the protein scaffold, an acylase gene optimized for expression in Escherichia coli and for molecular biology manipulations was designed.

Glycine oxidase from Bacillus subtilis. Characterization of a new flavoprotein.

Glycine oxidase (GO) is a homotetrameric flavoenzyme that contains one molecule of non-covalently bound flavin adenine dinucleotide per 47 kDa protein monomer. GO is active on various amines (sarcosine, N-ethylglycine, glycine) and d-amino acids (d-alanine, d-proline). The products of GO reaction with various substrates have been determined, and it has been clearly shown that GO catalyzes the oxidative deamination of primary and secondary amines, a reaction similar to that of d-amino acid oxidase, although its sequence homology is higher with enzymes such as sarcosine oxidase and N-methyltryptophane oxidase.