The structure of glucose-1-phosphate thymidylyltransferase from Mycobacterium tuberculosis reveals the location of an essential magnesium ion in the RmlA-type enzymes.

Tuberculosis, caused by the bacterium Mycobacterium tuberculosis, continues to be a major threat to populations worldwide. Whereas the disease is treatable, the drug regimen is arduous at best with the use of four antimicrobials over a six-month period. There is clearly a pressing need for the development of new therapeutics.

Structural and Biochemical Investigation of PglF from Campylobacter jejuni Reveals a New Mechanism for a Member of the Short Chain Dehydrogenase/Reductase Superfamily.

Within recent years it has become apparent that protein glycosylation is not limited to eukaryotes. Indeed, in Campylobacter jejuni, a Gram-negative bacterium, more than 60 of its proteins are known to be glycosylated. One of the sugars found in such glycosylated proteins is 2,4-diacetamido-2,4,6-trideoxy-α-d-glucopyranose, hereafter referred to as QuiNAc4NAc.

Biochemical Characterization of WbkC, an N-Formyltransferase from Brucella melitensis.

It has become increasingly apparent within the last several years that unusual N-formylated sugars are often found on the O-antigens of such Gram negative pathogenic organisms as Francisella tularensis, Campylobacter jejuni, and Providencia alcalifaciens, among others. Indeed, in some species of Brucella, for example, the O-antigen contains 1,2-linked 4-formamido-4,6-dideoxy-α-d-mannosyl groups. These sugars, often referred to as N-formylperosamine, are synthesized in pathways initiating with GDP-mannose.

Structural Studies on a Glucosamine/Glucosaminide N-Acetyltransferase.

Glucosamine/glucosaminide N-acetyltransferase or GlmA catalyzes the transfer of an acetyl group from acetyl CoA to the primary amino group of glucosamine. The enzyme from Clostridium acetobutylicum is thought to be involved in cell wall rescue. In addition to glucosamine, GlmA has been shown to function on di- and trisaccharides of glucosamine as well.

Examining Reaction Specificity in PvcB, a Source of Diversity in Isonitrile-Containing Natural Products.

Many bacteria produce isonitrile-containing natural products that are derived from aromatic amino acids. The synthetic clusters that control biosynthesis most commonly encode two enzymes, designated PvcA and PvcB, as well as additional enzymes that direct synthesis of the natural product. The PvcA enzyme installs the isonitrile moiety at the amino group of either tyrosine or tryptophan, as dictated by the particular pathway.

Evolutionary history and metabolic insights of ancient mammalian uricases.

Uricase is an enzyme involved in purine catabolism and is found in all three domains of life. Curiously, uricase is not functional in some organisms despite its role in converting highly insoluble uric acid into 5-hydroxyisourate. Of particular interest is the observation that apes, including humans, cannot oxidize uric acid, and it appears that multiple, independent evolutionary events led to the silencing or pseudogenization of the uricase gene in ancestral apes.

Structural and functional characterization of Pseudomonas aeruginosa AlgX: role of AlgX in alginate acetylation.

The exopolysaccharide alginate, produced by mucoid Pseudomonas aeruginosa in the lungs of cystic fibrosis patients, undergoes two different chemical modifications as it is synthesized that alter the properties of the polymer and hence the biofilm. One modification, acetylation, causes the cells in the biofilm to adhere better to lung epithelium, form microcolonies, and resist the effects of the host immune system and/or antibiotics. Alginate biosynthesis requires 12 proteins encoded by the algD operon, including AlgX, and although this protein is essential for polymer production, its exact role is unknown.

Functional characterization of AlgL, an alginate lyase from Pseudomonas aeruginosa.

Alginate lyase (AlgL) catalyzes the cleavage of the polysaccharide alginate through a β-elimination reaction. In Pseudomonas aeruginosa, algL is part of the alginate biosynthetic operon, and although it is required for alginate biosynthesis, it is not clear why. Steady-state kinetic studies were performed to characterize its substrate specificity and revealed that AlgL operates preferentially on nonacetylated alginate or its precursor mannuronan.

The enzymes of bacterial census and censorship.

N-Acyl-L-homoserine lactones (AHLs) are a major class of quorum-sensing signals used by Gram-negative bacteria to regulate gene expression in a population-dependent manner, thereby enabling group behavior. Enzymes capable of generating and catabolizing AHL signals are of significant interest for the study of microbial ecology and quorum-sensing pathways, for understanding the systems that bacteria have evolved to interact with small-molecule signals, and for their possible use in therapeutic and industrial applications. The recent structural and functional studies reviewed here provide a detailed insight into the chemistry and enzymology of bacterial communication.

Kinetic consequences of replacing the internucleotide phosphorus atoms in DNA with arsenic.

It was claimed in a recent publication that a strain of Halomonadacea bacteria (GFAJ-1) isolated from the arsenic-rich waters of Mono Lake, California is able to substitute arsenic for phosphorus in its macromolecules and small molecule metabolites. In this short Perspective, we consider chemical and biochemical issues surrounding the central claim that Halomonadacea GFAJ-1 is able to survive while incorporating kinetically labile arsenodiester linkages into the backbone of its DNA. Chemical precedents suggest that arsenodiester linkages in the putative arsenic-containing DNA of GFAJ-1 would undergo very rapid hydrolytic cleavage in water at 25 °C with an estimated half-life of 0.

Expression, purification, crystallization and preliminary X-ray analysis of Pseudomonas aeruginosa AlgX.

AlgX is a periplasmic protein required for the production of the exopolysaccharide alginate in Pseudomonas sp. and Azotobacter vinelandii. AlgX has been overexpressed and purified and diffraction-quality crystals have been grown using iterative seeding and the hanging-drop vapor-diffusion method.

Reactivity and reaction order in acylhomoserine lactone formation by Pseudomonas aeruginosa RhlI.

The formation of N-butyrylhomoserine lactone catalyzed by RhlI has been investigated by transient-state kinetic methods. A single intermediate, assigned to N-butyryl- S-adenosylmethionine, was observed. Under single-turnover conditions, the intermediate formed with a rate constant of 4.

Chemical mechanism and specificity of the C5-mannuronan epimerase reaction.

C5-mannuronan epimerase catalyzes the formation of alpha-L-guluronate residues from beta-D-mannuronate residues in the synthesis of the linear polysaccharide alginate. The reaction requires the abstraction of a proton from C5 of the residue undergoing epimerization followed by re-protonation on the opposite face. Rapid-mixing chemical quench experiments were conducted to determine the nature of the intermediate formed upon proton abstraction in the reaction catalyzed by the enzyme from Pseudomonas aeruginosa.

Pseudomonas aeruginosa C5-mannuronan epimerase: steady-state kinetics and characterization of the product.

Alginate is a major constituent of mature biofilms produced by Pseudomonas aeruginosa. The penultimate step in the biosynthesis of alginate is the conversion of some beta-D-mannuronate residues in the polymeric substrate polymannuronan to alpha-L-guluronate residues in a reaction catalyzed by C5-mannuronan epimerase. Specificity studies conducted with size-fractionated oligomannuronates revealed that the minimal substrate contained nine monosaccharide residues.

Update on ureide degradation in legumes.

Warm season N2-fixing legumes move fixed N from the nodules to the aerial portions of the plant primarily in the form of ureides, allantoin and allantoate, oxidation products of purines synthesized de novo in the nodule. Ureides are also products of purine turnover in senescing tissues, such as seedling cotyledons. A combination of biochemical and molecular approaches in both crop and model species has shed new light on the metabolic pathways involved in both the synthesis and degradation of allantoin.

Procatalytic ligand strain. Ionization and perturbation of 8-nitroxanthine at the urate oxidase active site.

The binding of the inhibitor 8-nitroxanthine to urate oxidase has been investigated by Raman and UV-visible absorption spectroscopy. The absorption maximum of 8-nitroxanthine shifts from 380 to 400 nm upon binding to the enzyme, demonstrating that the electronic structure of the ligand is perturbed. It has been proposed that oxidation of the substrate urate by urate oxidase is facilitated by formation of the substrate dianion at the enzyme active site, and Raman spectra of urate oxidase-bound 8-nitroxanthine suggest that both the dianionic and monoanionic forms of the ligand are bound to the enzyme under conditions where in solution the monoanion is present exclusively.

Inactivation of GDP-mannose dehydrogenase from Pseudomonas aeruginosa by penicillic acid identifies a critical active site loop.

The pathogenic bacterium Pseudomonas aeruginosa synthesizes alginate as one of a group of virulence factors that are produced during infections. The enzyme GDP-mannose dehydrogenase catalyzes the committed step in alginate biosynthesis. We show here that penicillic acid is an irreversible inactivator of GDP-mannose dehydrogenase.

Formation and reorientation of glucose 1,6-bisphosphate in the PMM/PGM reaction: transient-state kinetic studies.

The interconversion of glucose 1-phosphate and glucose 6-phosphate, catalyzed by Pseudomonas aeruginosa phosphomannomutase/phosphoglucomutase, has been studied by transient-state kinetic techniques. Glucose 1,6-bisphosphate is formed as an intermediate in the reaction, but an obligatory step in the catalytic cycle appears to be the formation of an enzyme-glucose 1,6-bisphosphate complex that is not competent to form either glucose 1-phosphate or glucose 6-phosphate directly. We suggest that during the lifetime of this complex the glucose 1,6-bisphosphate intermediate undergoes the 180 degrees reorientation that is required for completion of the catalytic cycle.

Chemical mechanism and substrate specificity of RhlI, an acylhomoserine lactone synthase from Pseudomonas aeruginosa.

The enzyme RhlI catalyzes the formation of N-butyrylhomoserine lactone from S-adenosylmethionine and N-butyrylacyl carrier protein. N-Butyrylhomoserine lactone serves as a quorum-sensing signal molecule in Pseudomonas aeruginosa, and is implicated in the regulation of many processes involved in bacterial virulence and infectivity. The P.

Detailed kinetic studies of an aggregating inhibitor; inhibition of phosphomannomutase/phosphoglucomutase by disperse blue 56.

Phosphomannomutase/phosphoglucomutase occupies a central position in the pathways by which several virulence factors are synthesized in Pseudomonas aeruginosa. Virtual screening was used to identify potential inhibitors of phosphomannomutase/ phosphoglucomutase, and one compound, the anthraquinone-based dye Disperse Blue 56, showed potent inhibition in vitro. The kinetics of inhibition was complex; the time courses for reactions in the presence of the inhibitor were biphasic, suggestive of slow-binding inhibition.

Structural basis of diverse substrate recognition by the enzyme PMM/PGM from P. aeruginosa.

Enzyme-substrate complexes of phosphomannomutase/phosphoglucomutase (PMM/PGM) reveal the structural basis of the enzyme's ability to use four different substrates in catalysis. High-resolution structures with glucose 1-phosphate, glucose 6-phosphate, mannose 1-phosphate, and mannose 6-phosphate show that the position of the phosphate group of each substrate is held constant by a conserved network of hydrogen bonds. This produces two distinct, and mutually exclusive, binding orientations for the sugar rings of the 1-phospho and 6-phospho sugars.

Roles of active site residues in Pseudomonas aeruginosa phosphomannomutase/phosphoglucomutase.

In Pseudomonas aeruginosa, the dual-specificity enzyme phosphomannomutase/phosphoglucomutase catalyzes the transfer of a phosphoryl group from serine 108 to the hydroxyl group at the 1-position of the substrate, either mannose 6-P or glucose 6-P. The enzyme must then catalyze transfer of the phosphoryl group on the 6-position of the substrate back to the enzyme. Each phosphoryl transfer is expected to require general acid-base catalysis, provided by amino acid residues at the enzyme active site.