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Report is presented on the binary complex structures of enzyme PrnB with D and L-tryptophan and D and L-7-chloro-tryptophan. The structures identify a common hydrophobic pocket for the indole ring but exhibit unusual heme ligation and substrate binding when compared with the tryptophan 2,3-dioxygenase crystal structures.

The structure of Streptococcus suis serotype type 2 dTDP-D-glucose 4,6-dehydratase (RmlB) is determined to 1.5 A resolution with its nicotinamide coenzyme and substrate analogue dTDP-xylose bound in and abortive complex. Results indicate the ability of dehydratase enzymes to fine-tune the redox potential of NADH through conformational changes in the nicotinamide ring.

The article discusses the mechanisms involved in the identification and evolution of the fluorination enzyme from Streptomyces cattleya. The enzyme is found to be widely used for catalyzing the C-F bond formation reactions.

The enzyme GDP-mannose-3',5'-epimerase (GME), which has the classical extended short-chain dehydratase/reductase (SDR) fold, carries out oxidation, epimerization and reduction in a single active site, yet preserves substrate selectivity. It is able to accomplish two epimerizations using the same acid/base pair of residues.

Research discusses biochemical transformations in terms of the enzymatic modification of sugar nucleotides with respect to both secondary metabolite glycosylation and the formation of cell surface-associated glycoconjugates in bacteria.

In a thermodynamic analysis of the UDP-galactopyranose mutase, it is seen that the enzyme substrate stabilizes a neutral flavosemiquinone radical with the production of the fully reduced flavin in an anionic form. Furthermore, the anionic flavohydroquinone facilitates rapid single electron transfer in a crypto-redox reaction.

The chemical structure of the amavadin form of vanadium is studied. Cyclic voltammetry are performed on amavadin and its chemical analogs by hydrogen nuclear magnetic resonance spectrum analysis at -280 ppm. Results reveal that the amavadin shares the same geometrical structure with its chemical analogs. The findings also indicate that amavadin belongs to the group of transition metals in biology which retain their structure through a one-electron redox reaction.

Research is presented describing the synthesis of a series of multivalent dendritic saccharides. A biophysical evaluation of their interaction with the plant lectin concanavalin A and strategies for overcoming 'weak binding' are discussed.

Enterobactin (ENT) is a siderophore (iron-chelating compound) produced by Escherichia coli to gain access to iron, an indispensable nutrient for bacterial growth. ENT is used as an exosiderophore by Pseudomonas aeruginosa with transport of ferri-ENT across the outer membrane by the PfeA transporter. Next to the pfeA gene on the chromosome is localized a gene encoding for an esterase, PfeE, whose transcription is regulated, as for pfeA, by the presence of ENT in bacterial environment. Purified PfeE hydrolyzed ferri-ENT into three molecules of 2,3-DHBS (2,3-dihydroxybenzoylserine) still complexed with ferric iron, and complete dissociation of iron from ENT chelating groups was only possible in the presence of both PfeE and an iron reducer, such as DTT. The crystal structure of PfeE and an inactive PfeE mutant complexed with ferri-ENT or a nonhydrolyzable ferri-catechol complex allowed identification of the enzyme binding site and the catalytic triad. Finally, cell fractionation and fluorescence microscopy showed periplasmic localization of PfeE in P. aeruginosa cells. Thus, the molecular mechanism of iron dissociation from ENT in P. aeruginosa differs from that previously described in E. coli. In P. aeruginosa, siderophore hydrolysis occurs in the periplasm, with ENT never reaching the bacterial cytoplasm. In E. coli, ferri-ENT crosses the inner membrane via the ABC transporter FepBCD and ferri-ENT is hydrolyzed by the esterase Fes only once it is in the cytoplasm.

Cyclic ribosomally derived peptides possess diverse bioactivities and are currently of major interest in drug development. However, it can be chemically challenging to synthesize these molecules, hindering the diversification and testing of cyclic peptide leads. Enzymes used in vitro offer a solution to this; however peptide macrocyclization remains the bottleneck. PCY1, involved in the biosynthesis of plant orbitides, belongs to the class of prolyl oligopeptidases and natively displays substrate promiscuity. PCY1 is a promising candidate for in vitro utilization, but its substrates require an 11 to 16 residue C-terminal recognition tail. We have characterized PCY1 both kinetically and structurally with multiple substrate complexes revealing the molecular basis of recognition and catalysis. Using these insights, we have identified a three residue C-terminal extension that replaces the natural recognition tail permitting PCY1 to operate on synthetic substrates. We demonstrate that PCY1 can macrocyclize a variety of substrates with this short tail, including unnatural amino acids and nonamino acids, highlighting PCY1's potential in biocatalysis.

3-Oxo-acyl-acyl carrier protein (ACP) reductase (FabG) plays a key role in the bacterial fatty acid synthesis II system in pathogenic microorganisms, which has been recognized as a potential drug target. FabG catalyzes reduction of a 3-oxo-acyl-ACP intermediate during the elongation cycle of fatty acid biosynthesis. Here, we report gene deletion experiments that support the essentiality of this gene in P. aeruginosa and the identification of a number of small molecule FabG inhibitors with IC50 values in the nanomolar to low micromolar range and good physicochemical properties. Structural characterization of 16 FabG-inhibitor complexes by X-ray crystallography revealed that the compounds bind at a novel allosteric site located at the FabG subunit-subunit interface. Inhibitor binding relies primarily on hydrophobic interactions, but specific hydrogen bonds are also observed. Importantly, the binding cavity is formed upon complex formation and therefore would not be recognized by virtual screening approaches. The structure analysis further reveals that the inhibitors act by inducing conformational changes that propagate to the active site, resulting in a displacement of the catalytic triad and the inability to bind NADPH.

Glucose-1-phosphate thymidylyltransferase (RmlA) catalyzes the condensation of glucose-1-phosphate (G1P) with deoxy-thymidine triphosphate (dTTP) to yield dTDP-d-glucose and pyrophosphate. This is the first step in the l-rhamnose biosynthetic pathway. l-Rhamnose is an important component of the cell wall of many microorganisms, including Mycobacterium tuberculosis and Pseudomonas aeruginosa. Here we describe the first nanomolar inhibitors of P. aeruginosa RmlA. These thymine analogues were identified by high-throughput screening and subsequently optimized by a combination of protein crystallography, in silico screening, and synthetic chemistry. Some of the inhibitors show inhibitory activity against M. tuberculosis. The inhibitors do not bind at the active site of RmlA but bind at a second site remote from the active site. Despite this, the compounds act as competitive inhibitors of G1P but with high cooperativity. This novel behavior was probed by structural analysis, which suggests that the inhibitors work by preventing RmlA from undergoing the conformational change key to its ordered bi-bi mechanism.

Recently a fluorination enzyme was identified and isolated from Streptomyces cattleya, as the first committed step on the metabolic pathway to the fluorinated metabolites, fluoroacetate and 4-fluorothreonine. This enzyme, 5'-fluoro-5'-deoxy adenosine synthetase (FDAS), has been shown to catalyze C-F bond formation by nucleophilic attack of fluoride ion to S-adenosyl-l-methionine (SAM) with the concomitant displacement of l-methionine to generate 5'-fluoro-5'-deoxy adenosine (5'-FDA). Although the structures of FDAS bound to both SAM and products have been solved, the molecular mechanism remained to be elucidated. We now report site-directed mutagenesis studies, structural analyses, and isothermal calorimetry (ITC) experiments. The data establish the key residues required for catalysis and the order of substrate binding. Fluoride ion is not readily distinguished from water by protein X-ray crystallography; however, using chloride ion (also a substrate) with a mutant of low activity has enabled the halide ion to be located in nonproductive co-complexes with SAH and SAM. The kinetic data suggest the positively charged sulfur of SAM is a key requirement in stabilizing the transition state. We propose a molecular mechanism for FDAS in which fluoride weakly associates with the enzyme exchanging two water molecules for protein ligation. The binding of SAM expels remaining water associated with fluoride ion and traps the ion in a pocket positioned to react with SAM, generating l-methionine and 5'-FDA. l-methionine then dissociates from the enzyme followed by 5'-FDA.