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The efficient inhibition of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) by the broad-spectrum herbicide glyphosate validates the shikimate pathway as a promising target for developing antimicrobial, fungicidal and herbicidal agents. The last enzyme of this pathway, chorismate synthase (CS), catalyses an unusual reaction, making it an attractive target for novel inhibitors. Therefore, we tested a series of azo-dyes for their inhibitory potential against CS from the pathogenic fungus Paracoccidioides brasiliensis ( Pb CS) and identified the azo-dye PH011669 that exhibits a dissociation ( K d ) and 50% inhibitory constant (IC 50 ) of 1.1 ± 0.1 and 10 ± 1 µM, respectively. Molecular docking and MD simulations provided insight into the mode of inhibition, showing that PH011669 binds to the enzyme's active site primarily through electrostatic interactions. Thus, our study is the first to integrate structural and computational methods to guide future efforts towards designing the next generation of CS inhibitors.

Chorismate serves as a crucial precursor for the synthesis of many aromatic compounds essential for the survival and virulence in various bacteria and protozoans. Chorismate synthase, a vital enzyme in the shikimate pathway, is responsible for the formation of chorismate from enolpyruvylshikimate-3-phosphate (EPSP). Moraxella catarrhalis is reported to be resistant to many beta-lactam antibiotics and causes chronic ailments such as otitis media, sinusitis, laryngitis, and bronchopulmonary infections. Here, we have cloned the aroC gene from Moraxella catarrhalis in pET28c and heterologously produced the chorismate synthase (~ 43 kDa) in Escherichia coli BL21(DE3) cells. We have predicted the three-dimensional structure of this enzyme and used the refined model for ligand-based virtual screening against Supernatural Database using PyRx tool that led to the identification of the top three molecules (caffeic acid, gallic acid, and o-coumaric acid). The resultant protein-ligand complex structures were subjected to 50 ns molecular dynamics (MD) simulation using GROMACS. Further, the binding energy was calculated by MM/PBSA approach using the trajectory obtained from MD simulation. The binding affinities of these compounds were validated with ITC experiments, which suggest that gallic acid has the highest binding affinity amongst these three phytochemicals. Together, these results pave the way for the use of these phytochemicals as potential anti-bacterial compounds.

Chlorogenic acid (CGA) is a phenolic compound with well-known antibacterial properties against pathogens. In this study, structural and biochemical characterization was used to show the inhibitory role of CGA against the enzyme of the shikimate pathway, a well-characterized drug target in several pathogens. Here, we report the crystal structures of dehydroquinate synthase (DHQS), the second enzyme of the shikimate pathway, from Providencia alcalifaciens ( Pa DHQS), in binary complex with NAD and ternary complex with NAD and CGA. Structural analyses reveal that CGA occupies the substrate position in the active site of Pa DHQS, which disables domain movements, leaving the enzyme in an open and catalysis-incompetent state. The binding analyses by isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) show that CGA binds to Pa DHQS with K D (equilibrium dissociation constant) values of 6.3 μM and 0.5 μM, respectively. In vitro e nzyme inhibition studies show that CGA inhibits Pa DHQS with a K i of 235 ± 21 μM, while it inhibits the growth of Providencia alcalifaciens , Moraxella catarrhalis , Staphylococcus aureus , and Escherichia coli with MIC values of 60 to 100 μM. In the presence of aromatic amino acids supplied externally, CGA does not show the toxic effect. These results, along with the observations of the inhibition of the 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) regulatory domain by CGA in our previous study, suggest that CGA binds to shikimate pathway enzymes with high affinity and inhibits their catalysis and can be further exploited for designing novel drug-like molecules. IMPORTANCE The shikimate pathway is an attractive target for the development of herbicides and antimicrobial agents, as it is essential in plants, bacteria, and apicomplexan parasites but absent in humans. The enzymes of shikimate pathway are conserved among bacteria. Thus, the inhibitors of the shikimate pathway act on wide range of pathogens. We have identified that chlorogenic acid targets the enzymes of the shikimate pathway. The crystal structure of dehydroquinate synthase, the second enzyme of the pathway, in complex with chlorogenic acid and enzymatic inhibition studies explains the mechanism of inhibition of chlorogenic acid. These results suggest that chlorogenic acid has a good chemical scaffold and have important implications for its further development as a potent inhibitor of shikimate pathway enzymes., (Copyright © 2020 American Society for Microbiology.)

Aim: A structural model of chorismate synthase (CS) from the pathogenic fungus Candida albicans was used for virtual screening simulations. Methods: Docking, molecular dynamics, cell growth inhibition and protein binding assays were used for search and validation. Results: Two molecules termed CS8 and CaCS02 were identified. Further studies of the minimal inhibitory concentration demonstrated fungicidal activity against Paracoccidioides brasiliensis with a minimal inhibitory concentration and minimal fungicidal concentration of 512 and 32 μg·ml -1 for CS8 and CaCS02, respectively. In addition, CaCS02 showed a strong synergistic effect in combination with amphotericin B without cytotoxic effects. In vitro studies using recombinant CS from P. brasiliensis showed IC 50 of 29 μM for CaCS02 supporting our interpretation that inhibition of CS causes the observed fungicidal activity.

Antimetabolites are small molecules that inhibit enzymes by mimicking physiological substrates. We report the discovery and structural elucidation of the antimetabolite 7-deoxy-sedoheptulose (7dSh). This unusual sugar inhibits the growth of various prototrophic organisms, including species of cyanobacteria, Saccharomyces, and Arabidopsis. We isolate bioactive 7dSh from culture supernatants of the cyanobacterium Synechococcus elongatus. A chemoenzymatic synthesis of 7dSh using S. elongatus transketolase as catalyst and 5-deoxy-D-ribose as substrate allows antimicrobial and herbicidal bioprofiling. Organisms treated with 7dSh accumulate 3-deoxy-D-arabino-heptulosonate 7-phosphate, which indicates that the molecular target is 3-dehydroquinate synthase, a key enzyme of the shikimate pathway, which is absent in humans and animals. The herbicidal activity of 7dSh is in the low micromolar range. No cytotoxic effects on mammalian cells have been observed. We propose that the in vivo inhibition of the shikimate pathway makes 7dSh a natural antimicrobial and herbicidal agent.

The bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP) is a key regulator of cellular motility, the cell cycle, and biofilm formation with its resultant antibiotic tolerance, which can make chronic infections difficult to treat. Therefore, diguanylate cyclases, which regulate the spatiotemporal production of c-di-GMP, might be attractive drug targets for control of biofilm formation that is part of chronic infections. We present a FRET-based biochemical high-throughput screening approach coupled with detailed structure-activity studies to identify synthetic small-molecule modulators of the diguanylate cyclase DgcA from Caulobacter crescentus. We identified a set of seven small molecules that regulate DgcA enzymatic activity in the low-micromolar range. Subsequent structure-activity studies on selected scaffolds revealed a remarkable diversity of modulatory behavior, including slight chemical substitutions that reverse the effects from allosteric enzyme inhibition to activation. The compounds identified represent new chemotypes and are potentially developable into chemical genetic tools for the dissection of c-di-GMP signaling networks and alteration of c-di-GMP-associated phenotypes. In sum, our studies underline the importance of detailed mechanism-of-action studies for inhibitors of c-di-GMP signaling and demonstrate the complex interplay between synthetic small molecules and the regulatory mechanisms that control the activity of diguanylate cyclases., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Paracoccidioidomycosis (PCM), caused by Paracoccidioides , is a systemic mycosis with granulomatous character and a restricted therapeutic arsenal. The aim of this work was to search for new alternatives to treat largely neglected tropical mycosis, such as PCM. In this context, the enzymes of the shikimate pathway constitute excellent drug targets for conferring selective toxicity because this pathway is absent in humans but essential for the fungus. In this work, we have used a homology model of the chorismate synthase (EC 4.2.3.5) from Paracoccidioides brasiliensis ( Pb CS) and performed a combination of virtual screening and molecular dynamics testing to identify new potential inhibitors. The best hit, CP1, successfully adhered to pharmacological criteria (adsorption, distribution, metabolism, excretion, and toxicity) and was therefore used in in vitro experiments. Here we demonstrate that CP1 binds with a dissociation constant of 64 ± 1 μM to recombinant chorismate synthase from P. brasiliensis and inhibits enzymatic activity, with a 50% inhibitory concentration (IC 50 ) of 47 ± 5 μM. As expected, CP1 showed no toxicity in three cell lines. On the other hand, CP1 reduced the fungal burden in lungs from treated mice, similar to itraconazole. In addition, histopathological analysis showed that animals treated with CP1 displayed less lung tissue infiltration, fewer yeast cells, and large areas with preserved architecture. Therefore, CP1 was able to control PCM in mice with a lower inflammatory response and is thus a promising candidate and lead structure for the development of drugs useful in PCM treatment., (Copyright © 2018 American Society for Microbiology.)

Shikimate pathway plays an essential role in the biosynthesis of aromatic amino acids in various plants and bacteria, which consists of seven key enzymes and they are all attractive targets for antibacterial agent development due to their absence in humans. The Staphylococcus aureus dehydroquinate synthase (SaDHQS) is involved in the second step of shikimate pathway, which catalyzes the NAD + -dependent conversion of 3-deoxy-D-arabino-heptulosonate-7-phosphate to dehydroquinate via multiple steps. The enzyme active site can be characterized by two spatially separated subpockets 1 and 2, which represent the reaction center of substrate adduct with NAD + nicotinamide moiety and the assistant binding site of NAD + adenine moiety, respectively. In silico virtual screening is performed against a biogenic compound library to discover SaDHQS subpocket-specific inhibitors, which were then tested against both antibiotic-sensitive and antibiotic-resistant S. aureus strains by using in vitro susceptibility test. The activity profile of hit compounds has no considerable difference between the antibiotic-sensitive and -resistant strains. The subpocket 1-specific inhibitors exhibit a generally higher activity than subpocket 2-specific inhibitors, and they also hold a strong selectivity between their cognate and noncognate subpockets. Dynamics and energetics analyses reveal that the SaDHQS active site prefers to interact with amphipathic and polar inhibitors by forming multiple hydrogen bonds and van der Waals packing at the complex interfaces of the two subpockets with their cognate inhibitors.

The anti-tuberculosis (TB) agent IMB-T130 was speculated to be a multi-target compound. In this research, we found that IMB-T130 inhibits the catalytic activity of Mycobacterium tuberculosis 3-dehydroquinate synthase (MtDHQS), the enzyme in the second step of the shikimate pathway. IMB-T130 was identified as a selective inhibitor of MtDHQS with an IC 50 value of 0.87 μg/mL. The interaction between the compound and protein was analysed by surface plasmon resonance and circular dichroism. Based on the in silico molecular docking results, the essential amino acids in the binding pocket were then confirmed by site-directed mutagenesis. Overexpression of DHQS reduced the antibacterial activity of IMB-T130 in cells, verifying that DHQS is the target of IMB-T130. IMB-T130 inhibited standard and drug-resistant M. tuberculosis strains by targeting DHQS. Our findings improve our understanding of MtDHQS and make it to be a potential target for new anti-TB drug discovery.

One of the most important signals involved in controlling biofilm formation is represented by the intracellular second messenger 3',5'-cyclic diguanylic acid (c-di-GMP). Since the pathways involved in c-di-GMP biosynthesis and breakdown are found only in bacteria, targeting c-di-GMP metabolism represents an attractive strategy for the development of biofilm-disrupting drugs. Here, we present the workflow required to perform a structure-based design of inhibitors of diguanylate cyclases, the enzymes responsible for c-di-GMP biosynthesis. Downstream of the virtual screening process, detailed in the first part of the chapter, we report the step-by-step protocols required to test the positive hits in vitro and to validate their selectivity, thus minimizing possible off-target effects.

Bacteria can sense environmental cues and alter their physiology accordingly through the use of signal transduction pathways involving second messenger nucleotides. One broadly conserved second messenger is cyclic-di-AMP (c-di-AMP) which regulates a range of processes including cell wall homeostasis, potassium uptake, DNA repair, fatty acid synthesis, biofilm formation and central metabolism in bacteria. The intracellular pool of c-di-AMP is maintained by the activities of diadenylate cyclase (DAC) and phosphodiesterase (PDE) enzymes, as well as possibly via c-di-AMP export. Whilst extracellular stimuli regulating c-di-AMP levels in bacteria are poorly understood, recent work has identified effector proteins which directly interact and alter the activity of DACs. These include the membrane bound CdaR and the phosphoglucosamine mutase GlmM which both bind directly to the membrane bound CdaA DAC and the recombination protein RadA which binds directly to the DNA binding DisA DAC. The genes encoding these multiprotein complexes are co-localised in many bacteria providing further support for their functional connection. The roles of GlmM in peptidoglycan synthesis and RadA in Holliday junction intermediate processing suggest that c-di-AMP synthesis by DACs will be responsive to these cellular activities. In addition to these modulatory interactions, permanent dysregulation of DAC activity due to suppressor mutations can occur during selection to overcome growth defects, rapid cell lysis and osmosensitivity. DACs have also been investigated as targets for the development of new antibiotics and several small compound inhibitors have recently been identified. This review aims to provide an overview of how c-di-AMP synthesis by DACs can be regulated.

Cyclic di-AMP has emerged as an important signaling molecule that controls a myriad of functions, including cell wall homeostasis in different bacteria. Polyphenols display various biological activities and tea polyphenols in particular have been shown to possess among other properties antioxidant and antibacterial activities. Certain tea polyphenols, such as catechin and epigallocatechin gallate, have been used to augment the action of traditional antibiotics that target the cell wall. Considering the expanding role played by cyclic dinucleotides in bacteria, we investigated whether the action of polyphenols on bacteria could be due in part to modulation of c-di-AMP signaling. Out of 14 tested polyphenols, tannic acid (TA), theaflavin-3'-gallate (TF2B) and theaflavin-3,3'-digallate (TF3) exhibited inhibitory effects on B. subtilis c-di-AMP synthase, DisA. TF2B and TF3 specifically inhibited DisA but not YybT (a PDE) whilst TA was more promiscuous and inhibited both DisA and YybT.

C-di-AMP synthases are essential in several bacteria, including human pathogens; hence these enzymes are potential antibiotic targets. However, there is a dearth of small molecule inhibitors of c-di-AMP metabolism enzymes. Screening of 2000 known drugs against DisA has led to the identification of suramin, an antiparasitic drug as potent inhibitor of c-di-AMP synthase.

2-Methylerythritol 2,4-cyclodiphosphate synthase (IspF) is an essential enzyme for the biosynthesis of isoprenoid precursors in plants and many human pathogens. The protein is an attractive target for the development of anti-infectives and herbicides. Using a photometric assay, a screen of 40 000 compounds on IspF from Arabidopsis thaliana afforded symmetrical aryl bis-sulfonamides that inhibit IspF from A. thaliana (AtIspF) and Plasmodium falciparum (PfIspF) with IC50 values in the micromolar range. The ortho-bis-sulfonamide structural motif is essential for inhibitory activity. The best derivatives obtained by parallel synthesis showed IC50 values of 1.4 μm against PfIspF and 240 nm against AtIspF. Substantial herbicidal activity was observed at a dose of 2 kg ha(-1) . Molecular modeling studies served as the basis for an in silico search targeted at the discovery of novel, non-symmetrical sulfonamide IspF inhibitors. The designed compounds were found to exhibit inhibitory activities in the double-digit micromolar IC50 range., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cyclic di-GMP (c-di-GMP) is a widespread second messenger that plays a key role in bacterial biofilm formation. The compound's ability to assume multiple conformations allows it to interact with a diverse set of target macromolecules. Here, we analyzed the binding mode of c-di-GMP to the allosteric inhibitory site (I-site) of diguanylate cyclases (DGCs) and compared it to the conformation adopted in the catalytic site of the EAL phosphodiesterases (PDEs). An array of novel molecules has been designed and synthesized by simplifying the native c-di-GMP structure and replacing the charged phosphodiester backbone with an isosteric nonhydrolyzable 1,2,3-triazole moiety. We developed the first neutral small molecule able to selectively target DGCs discriminating between the I-site of DGCs and the active site of PDEs; this molecule represents a novel tool for mechanistic studies, particularly on those proteins bearing both DGC and PDE modules, and for future optimization studies to target DGCs in vivo.

Unlabelled: Biofilm formation is responsible for increased antibiotic tolerance in pathogenic bacteria. Cyclic di-GMP (c-di-GMP) is a widely used second-messenger signal that plays a key role in bacterial biofilm formation. c-di-GMP is synthesized by diguanylate cyclases (DGCs), a conserved class of enzymes absent in mammals and hence considered attractive molecular targets for the development of antibiofilm agents. Here, the results of a virtual screening approach aimed at identifying small-molecule inhibitors of the DGC PleD from Caulobacter crescentus are described. A three-dimensional (3D) pharmacophore model, derived from the mode of binding of GTP to the active site of PleD, was exploited to screen the ZINC database of compounds. Seven virtual hits were tested in vitro for their ability to inhibit the activity of purified PleD by using circular dichroism spectroscopy. Two drug-like molecules with a catechol moiety and a sulfonohydrazide scaffold were shown to competitively inhibit PleD at the low-micromolar range (50% inhibitory concentration [IC50] of ∼11 μM). Their predicted binding mode highlighted key structural features presumably responsible for the efficient inhibition of PleD by both hits. These molecules represent the most potent in vitro inhibitors of PleD identified so far and could therefore result in useful leads for the development of novel classes of antimicrobials able to hamper biofilm formation., Importance: Biofilm-mediated infections are difficult to eradicate, posing a threatening health issue worldwide. The capability of bacteria to form biofilms is almost universally stimulated by the second messenger c-di-GMP. This evidence has boosted research in the last decade for the development of new antibiofilm strategies interfering with c-di-GMP metabolism. Here, two potent inhibitors of c-di-GMP synthesis have been identified in silico and characterized in vitro by using the well-characterized DGC enzyme PleD from C. crescentus as a structural template and molecular target. Given that the protein residues implied as crucial for enzyme inhibition are found to be highly conserved among DGCs, the outcome of this study could pave the way for the future development of broad-spectrum antibiofilm compounds., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Important pathogens such as Mycobacterium tuberculosis and Plasmodium falciparum, the causative agents of tuberculosis and malaria, respectively, and plants, utilize the 2C-methyl-D-erythritol 4-phosphate (MEP, 5) pathway for the biosynthesis of isopentenyl diphosphate (1) and dimethylallyl diphosphate (2), the universal precursors of isoprenoids, while humans exclusively utilize the alternative mevalonate pathway for the synthesis of 1 and 2. This distinct distribution, together with the fact that the MEP pathway is essential in numerous organisms, makes the enzymes of the MEP pathway attractive drug targets for the development of anti-infective agents and herbicides. Herein, we review the inhibitors reported over the past 2 years, in the context of the most important older developments and with a particular focus on the results obtained against enzymes of pathogenic organisms. We will also discuss new discoveries in terms of structural and mechanistic features, which can help to guide a rational development of inhibitors.

Large-scale production of bis-3'-5'-cyclic-di-GMP (c-di-GMP) would facilitate biological studies of numerous bacterial signaling pathways and phenotypes controlled by this second messenger molecule, such as virulence and biofilm formation. C-di-GMP constitutes also a potentially interesting molecule as a vaccine adjuvant. Even though chemical synthesis of c-di-GMP can be done, the yields are incompatible with mass-production. tDGC, a stand-alone diguanylate cyclase (DGC or GGDEF domain) from Thermotoga maritima, enables the robust enzymatic production of large quantities of c-di-GMP. To understand the structural correlates of tDGC thermostability, its catalytic mechanism and feedback inhibition, we determined structures of an active-like dimeric conformation with both active (A) sites facing each other and of an inactive dimeric conformation, locked by c-di-GMP bound at the inhibitory (I) site. We also report the structure of a single mutant of tDGC, with the R158A mutation at the I-site, abolishing product inhibition and unproductive dimerization. A comparison with structurally characterized DGC homologues from mesophiles reveals the presence of a higher number of salt bridges in the hyperthermophile enzyme tDGC. Denaturation experiments of mutants disrupting in turn each of the salt bridges unique to tDGC identified three salt-bridges critical to confer thermostability.

The rise of bacterial resistance to traditional antibiotics has motivated recent efforts to identify new drug candidates that target virulence factors or their regulatory pathways. One such antivirulence target is the cyclic-di-GMP (cdiGMP) signaling pathway, which regulates biofilm formation, motility, and pathogenesis. Pseudomonas aeruginosa is an important opportunistic pathogen that utilizes cdiGMP-regulated polysaccharides, including alginate and pellicle polysaccharide (PEL), to mediate virulence and antibiotic resistance. CdiGMP activates PEL and alginate biosynthesis by binding to specific receptors including PelD and Alg44. Mutations that abrogate cdiGMP binding to these receptors prevent polysaccharide production. Identification of small molecules that can inhibit cdiGMP binding to the allosteric sites on these proteins could mimic binding defective mutants and potentially reduce biofilm formation or alginate secretion. Here, we report the development of a rapid and quantitative high-throughput screen for inhibitors of protein-cdiGMP interactions based on the differential radial capillary action of ligand assay (DRaCALA). Using this approach, we identified ebselen as an inhibitor of cdiGMP binding to receptors containing an RxxD domain including PelD and diguanylate cyclases (DGC). Ebselen reduces diguanylate cyclase activity by covalently modifying cysteine residues. Ebselen oxide, the selenone analogue of ebselen, also inhibits cdiGMP binding through the same covalent mechanism. Ebselen and ebselen oxide inhibit cdiGMP regulation of biofilm formation and flagella-mediated motility in P. aeruginosa through inhibition of diguanylate cyclases. The identification of ebselen provides a proof-of-principle that a DRaCALA high-throughput screening approach can be used to identify bioactive agents that reverse regulation of cdiGMP signaling by targeting cdiGMP-binding domains.

Biofilm formation by pathogenic bacteria is an important virulence factor in the development of numerous chronic infections, thereby causing a severe health burden. Many of these infections cannot be resolved, as bacteria in biofilms are resistant to the host's immune defenses and antibiotic therapy. An urgent need for new strategies to treat biofilm-based infections is critically needed. Cyclic di-GMP (c-di-GMP) is a widely conserved second-messenger signal essential for biofilm formation. The absence of this signalling system in higher eukaryotes makes it an attractive target for the development of new anti-biofilm agents. In this study, the results of an in silico pharmacophore-based screen to identify small-molecule inhibitors of diguanylate cyclase (DGC) enzymes that synthesize c-di-GMP are described. Four small molecules, LP 3134, LP 3145, LP 4010 and LP 1062 that antagonize these enzymes and inhibit biofilm formation by Pseudomonas aeruginosa and Acinetobacter baumannii in a continuous-flow system are reported. All four molecules dispersed P. aeruginosa biofilms and inhibited biofilm development on urinary catheters. One molecule dispersed A. baumannii biofilms. Two molecules displayed no toxic effects on eukaryotic cells. These molecules represent the first compounds identified from an in silico screen that are able to inhibit DGC activity to prevent biofilm formation.

The methylerythritol phosphate (MEP) pathway of Plasmodium falciparum (P. falciparum) has become an attractive target for anti-malarial drug discovery. This study describes a kinetic model of this pathway, its use in validating 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) as drug target from the systemic perspective, and additional target identification, using metabolic control analysis and in silico inhibition studies. In addition to DXR, 1-deoxy-d-xylulose 5-phosphate synthase (DXS) can be targeted because it is the first enzyme of the pathway and has the highest flux control coefficient followed by that of DXR. In silico inhibition of both enzymes caused large decrement in the pathway flux. An added advantage of targeting DXS is its influence on vitamin B1 and B6 biosynthesis. Two more potential targets, 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase, were also identified. Their inhibition caused large accumulation of their substrates causing instability of the system. This study demonstrates that both types of enzyme targets, one acting via flux reduction and the other by metabolite accumulation, exist in P. falciparum MEP pathway. These groups of targets can be exploited for independent anti-malarial drugs., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Cyclic-di-GMP (c-di-GMP) is a central regulator of bacterial behavior. Various studies have implicated c-di-GMP in biofilm formation and virulence factor production in multitudes of bacteria. Hence it is expected that the disruption of c-di-GMP signaling could provide an effective means to disrupt biofilm and/or virulence factor formation in several bacteria of clinical relevance. C-di-GMP achieves the regulation of bacterial phenotype via binding to several effector molecules including transcription factors, enzymes and riboswitches. Crystal structure analyses of c-di-GMP effector molecules, in complex with the ligand, reveal that various classes of c-di-GMP receptors recognize this dinucleotide using different sets of recognition elements. Therefore, it is plausible that different analogues of c-di-GMP could be used to selectively modulate a specific class of c-di-GMP binding receptors, and hence modulate the bacterial phenotype. Thus far only a detailed study of the differential binding of c-di-GMP analogues to riboswitches, but not proteins, has been reported. In this report, we prepared various 2'-modified analogues of c-di-GMP and studied both polymorphisms of these analogues using DOSY NMR and the binding to several effector proteins, such as PilZ-containing proteins, diguanylate cyclases (DGC) containing I-sites, and phoshphodiesterases (PDE). 2'-Modification of c-di-GMP did not adversely affect the propensity to form higher aggregates, such as octameric forms, in the presence of potassium salts. Interestingly, we find that the selective binding to different classes of c-di-GMP binding proteins could be achieved with the 2'-modified analogues and that 2'-F analogue of c-di-GMP binds to the I-site of DGCs better (four times) than the native dinucleotide, c-di-GMP, whereas c-di-GMP binds to PDEs better (10 times) than 2'-F-c-di-GMP. 2'-F-c-di-GMP potently inhibits c-di-GMP synthesis by DGCs and hence raises the potential that cell permeable analogues of 2'-F-c-di-GMP could be used to disrupt c-di-GMP signaling in bacteria., (Copyright © 2013. Published by Elsevier Ltd.)

Diguanylate cyclases synthesize the second messenger c-di-GMP, which in turn governs a plethora of physiological processes in bacteria. Although most diguanylate cyclases harbor sensory domains, their input signals are largely unknown. Here, we demonstrate that diguanylate cyclase DgcZ (YdeH) from Escherichia coli is regulated allosterically by zinc. Crystal structures show that the zinc ion is bound to the 3His/1Cys motif of the regulatory chemoreceptor zinc-binding domain, which mediates subunit contact within the dimeric enzyme. In vitro, zinc reversibly inhibits DgcZ with a subfemtomolar Ki constant. In vivo, bacterial biofilm formation is modulated by externally applied zinc in a DgcZ- and c-di-GMP-dependent fashion. The study outlines the structural principles of this zinc sensor. Zinc binding seems to regulate the activity of the catalytic GGDEF domains by impeding their mobility and thus preventing productive encounter of the two GTP substrates., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: Infectious diseases are major public health problem. It is increasingly affecting more than 50 million people worldwide. Targeting shikimate pathway could be efficiently used for the development of broad spectrum antimicrobial compound against variety of infectious diseases. Chorismate synthase is an enzyme in shikimate pathway that catalyzes Phosphoenol pyruvate to chorismate in most of the prokaryotic bacteria. This step is crucial for its growth, since Chorismate acts as a precursor molecule for the synthesis of aromatic amino acids. Hence, we present a comprehensive database of Chorismate Synthase Database (CSDB) which is a manually curated database. It provides information on the sequence, structure and biological activity of chorismate synthase from shikimate pathway of pathogenic bacteria. Design of suitable inhibitors for this enzyme, hence could be a probable solution to destroy its proteomic machinery and thereby inhibit the bacterial growth., Description: The aim of this study was to characterise chorismate synthase enzyme belonging to pathogenic bacteria to analysis the functional and structural characterization of chorismate synthase is very important for both structure-based and ligand based drug design., Conclusions: The broad range of data easy to use user interface makes csdb.in a useful database for researchers in designing drugs.

Chorismatases and isochorismatases catalyse the hydrolysis of chorismate or isochorismate leading to unsaturated cyclohexenoic acid derivatives. Based on simplification of the physiological substrates, two cinnamic acid-derived compounds, differing in the saturation of the side chain, were developed. In contrast to earlier inhibitor studies, the compounds described here do not have an ether bond and therefore can be synthesised very easily in one or two steps without the need for protective groups. Both substances demonstrate inhibition of the isochorismatase EntB from Escherichia coli and the chorismatases FkbO and Hyg5 from Streptomyces. For chorismatases, the unsaturated compound shows IC(50) values in the millimolar range, while the saturated compound is the better inhibitor with IC(50) values in the micromolar/low millimolar range; for the isochorismatase tested both compounds inhibit in the micromolar range. Further, an analysis of the apparent K(m) values for FkbO and EntB was performed, showing that both inhibitors act in a competitive manner. Due to the ease of modifying these new inhibitors they are a suitable starting point for exploring further functionalised derivatives., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

There is significant progress toward understanding catalysis throughout the essential MEP pathway to isoprenoids in human pathogens; however, little is known about pathway regulation. The present study begins by testing the hypothesis that isoprenoid biosynthesis is regulated via feedback inhibition of the fifth enzyme cyclodiphosphate synthase IspF by downstream isoprenoid diphosphates. Here, we demonstrate recombinant E. coli IspF is not inhibited by downstream metabolites isopentenyl diphosphate (IDP), dimethylallyl diphosphate (DMADP), geranyl diphosphate (GDP), and farnesyl diphosphate (FDP) under standard assay conditions. However, 2C-methyl-d-erythritol 4-phosphate (MEP), the product of reductoisomerase IspC and first committed MEP pathway intermediate, activates and sustains this enhanced IspF activity, and the IspF-MEP complex is inhibited by FDP. We further show that the methylerythritol scaffold itself, which is unique to this pathway, drives the activation and stabilization of active IspF. Our results suggest a novel feed-forward regulatory mechanism for 2C-methyl-d-erythritol 2,4-cyclodiphosphate (MEcDP) production and support an isoprenoid biosynthesis regulatory mechanism via feedback inhibition of the IspF-MEP complex by FDP. The results have important implications for development of inhibitors against the IspF-MEP complex, which may be the physiologically relevant form of the enzyme.

Bacterial biofilm formation is responsible for numerous chronic infections, causing a severe health burden. Many of these infections cannot be resolved, as bacteria in biofilms are resistant to the host's immune defenses and antibiotic therapy. New strategies to treat biofilm-based infections are critically needed. Cyclic di-GMP (c-di-GMP) is a widely conserved second-messenger signal essential for biofilm formation. As this signaling system is found only in bacteria, it is an attractive target for the development of new antibiofilm interventions. Here, we describe the results of a high-throughput screen to identify small-molecule inhibitors of diguanylate cyclase (DGC) enzymes that synthesize c-di-GMP. We report seven small molecules that antagonize these enzymes and inhibit biofilm formation by Vibrio cholerae. Moreover, two of these compounds significantly reduce the total concentration of c-di-GMP in V. cholerae, one of which also inhibits biofilm formation by Pseudomonas aeruginosa in a continuous-flow system. These molecules represent the first compounds described that are able to inhibit DGC activity to prevent biofilm formation.

The shikimate pathway in Plasmodium falciparum provides several targets for designing novel antiparasitic agents for the treatment of malaria. Chorismate synthase (CS) is a key enzyme in the shikimate pathway which catalyzes the seventh and final step of the pathway. P. falciparum chorismate synthase (PfCS) is unique in terms of enzymatic behavior, cellular localization and in having two additional amino acid inserts compared to any other CS. The structure of PfCS along with cofactor FMN was predicted by homology modeling using crystal structure of Helicobacter pylori chorismate synthase (HpCS). The quality of the model was validated using structure analysis servers and molecular dynamics. Dimeric form of PfCS was generated and the FMN binding mechanism involving movement of loop near active site has been proposed. Active site pocket has been identified and substrate 5-enolpyruvylshikimate 3-phosphate (EPSP) along with screened potent inhibitors has been docked. The study resulted in identification of putative inhibitors of PfCS with binding efficiency in nanomolar range. The selected putative inhibitors could lead to the development of anti-malarial drugs., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We present a novel algorithm called CrystalDock that analyzes a molecular pocket of interest and identifies potential binding fragments. The program first identifies groups of pocket-lining receptor residues (i.e., microenvironments) and then searches for geometrically similar microenvironments present in publically available databases of ligand-bound experimental structures. Germane fragments from the crystallographic or NMR ligands are subsequently placed within the novel binding pocket. These positioned fragments can be linked together to produce ligands that are likely to be potent; alternatively, they can be joined to an inhibitor with a known or suspected binding pose to potentially improve binding affinity. To demonstrate the utility of the algorithm, CrystalDock is used to analyze the principal binding pockets of influenza neuraminidase and Trypanosoma brucei RNA editing ligase 1, validated drug targets in the fight against pandemic influenza and African sleeping sickness, respectively. In both cases, CrystalDock suggests modifications to known inhibitors that may improve binding affinity.

Tuberculosis is considered a worldwide health problem mainly due to co-infection with HIV and proliferation of multi-drug-resistant strains. The enzymes of the shikimate pathway are potential targets for the development of new therapies because they are essential for bacteria, but absent from mammals. The last step in this pathway is performed by chorismate synthase (CS), which catalyzes the conversion of 5-enolpyruvylshikimate-3-phosphate (EPSP) to chorismate. The aim of this article is to review the available information on chorismate synthase from Mycobacterium tuberculosis.

Structural genomics efforts have traditionally focused on generating single protein structures of unique and diverse targets. However, a lone structure for a given target is often insufficient to firmly assign function or to drive drug discovery. As part of the Seattle Structural Genomics Center for Infectious Disease (SSGCID), we seek to expand the focus of structural genomics by elucidating ensembles of structures that examine small molecule-protein interactions for selected infectious disease targets. In this chapter, we discuss two applications for small molecule libraries in structural genomics: unbiased fragment screening, to provide inspiration for lead development, and targeted, knowledge-based screening, to confirm or correct the functional annotation of a given gene product. This shift in emphasis results in a structural genomics effort that is more engaged with the infectious disease research community, and one that produces structures of greater utility to researchers interested in both protein function and inhibitor development. We also describe specific methods for conducting high-throughput fragment screening in a structural genomics context by X-ray crystallography., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Five analogs of cyclic di-nucleotidic acid including c-di-GMP were synthesized and evaluated for their biological activities on Slr1143, a diguanylate cyclase of Synechocystis sp. Slr1143 was overexpressed from the recombinant plasmid which contained the gene of interest and subsequently purified by affinity chromatography. A new HPLC method capable of separating the compound and product peaks with good resolution was optimized and applied to the analysis of the compounds. Results obtained show that cyclic di-inosinylic acid 1b demonstrates a stronger inhibition on Slr1143 than c-di-GMP and is a potential inhibitor for biofilm formation., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Background: The methylerythritol phosphate pathway for isoprenoid biosynthesis is an attractive target for the design of new specific antibiotics for the treatment of gastrointestinal diseases associated with the presence of the bacterium Helicobacter pylori since this pathway which is essential to the bacterium is absent in humans., Results: This work reports the molecular cloning of one of the genes of the methylerythritol phosphate pathway form H. pylori (ispDF; HP_1440) its expression in Escherichia coli and the functional characterization of the recombinant enzyme. As shown by genetic complementation and in vitro functional assays the product of the ispDF gene form H. pylori is a bifunctional enzyme which can replace both CDP-methylerythritol synthase and methylerythritol cyclodiphosphate synthase from E. coli., General Significance: Designing inhibitors that affect at the same time both enzyme activities of the H. pylori bifunctional enzyme (i.e. by disrupting protein oligomerization) would result in more effective antibiotics which would be able to continue their action even if the bacterium acquired a resistance to another antibiotic directed against one of the individual activities., Conclusion: The bifunctional enzyme would be an excellent target for the design of new, selective antibiotics for the treatment of H. pylori associated diseases., (Copyright © 2010 Elsevier B.V. All rights reserved.)

A library of 40,000 compounds was screened for inhibitors of 2-methylerythritol 2,4-cyclodiphosphate synthase (IspF) protein from Arabidopsis thaliana using a photometric assay. A thiazolopyrimidine derivative resulting from the high-throughput screen was found to inhibit the IspF proteins of Mycobacterium tuberculosis, Plasmodium falciparum, and A. thaliana with IC(50) values in the micromolar range. Synthetic efforts afforded derivatives that inhibit IspF protein from M. tuberculosis and P. falciparum with IC(50) values in the low micromolar range. Several compounds act as weak inhibitors in the P. falciparum red blood cell assay.

Class II diterpene cyclases mediate the acid-initiated cycloisomerization reaction that serves as the committed step in biosynthesis of the large class of labdane-related diterpenoid natural products, which includes the important gibberellin plant hormones. Intriguingly, these enzymes are differentially susceptible to inhibition by their Mg(2+) cofactor, with those involved in gibberellin biosynthesis being more sensitive to such inhibition than those devoted to secondary metabolism, which presumably limits flux toward the potent gibberellin phytohormones. Such inhibition has been suggested to arise from intrasteric Mg(2+) binding to the DXDD motif that cooperatively acts as the catalytic acid, whose affinity must then be modulated in some fashion. While further investigating class II diterpene cyclase catalysis, we discovered a conserved basic residue that seems to act as a counter ion to the DXDD motif, enhancing the ability of aspartic acid to carry out the requisite energetically difficult protonation of a carbon-carbon double bond and also affecting inhibitory Mg(2+) binding. Notably, this residue is conserved as a histidine in enzymes involved in gibberellin biosynthesis and as an arginine in those dedicated to secondary metabolism. Interchanging the identity of these residues is sufficient to switch the sensitivity of the parent enzyme to inhibition by Mg(2+). These striking findings indicate that this is a single residue switch for Mg(2+) inhibition, which not only supports the importance of this biochemical regulatory mechanism in limiting gibberellin biosynthesis, but the importance of its release, presumably to enable higher flux, into secondary metabolism.

In Gram-negative bacteria, production of bis-(3',5')-cyclic diguanylic acid (c-di-GMP) by diguanylate cyclases (DGCs) is the main trigger for production of extracellular polysaccharides and for biofilm formation. Mutants affected in c-di-GMP biosynthesis are impaired in biofilm formation, thus making DGCs interesting targets for new antimicrobial agents with anti-biofilm activity. In this report, we describe a strategy for the screening for DGC inhibitors consisting of a combination of three microbiological assays. The primary assay utilizes an Escherichia coli strain overexpressing the adrA gene, encoding the DGC protein AdrA, and relies on detection of AdrA-dependent cellulose production as red colony phenotype on solid medium supplemented with the dye Congo red (CR). Presence of DGC inhibitors blocking AdrA activity would result in a white phenotype on CR medium. The CR assay can be performed in 96-well microtiter plates, making it suitable for high-throughput screenings. To confirm specific inhibition of c-di-GMP biosynthesis, chemical compounds positive in the CR assay are tested for their ability to inhibit biofilm formation and in a reporter gene assay which monitors expression of curli-encoding genes as a function of DGC activity. Screening of a chemical library using the described approach allowed us to identify sulfathiazole, an antimetabolite drug, as an inhibitor of c-di-GMP biosynthesis. Sulfathiazole probably affects c-di-GMP biosynthesis in an indirect fashion rather than by binding to DGCs; however, sulfathiazole represents the first example of drug able to affect biofilm formation by interfering with c-di-GMP metabolism.

Dehydroquinate synthase (DHQS) is a nicotinamide adenine dinucleotide (NAD)-dependent enzyme that converts 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) into 3-dehydroquinate (DHQ). Since it catalyzes the second key step in the shikimate pathway, which is crucial for the aromatic amino acid metabolism in bacteria, fungi, and plants, but not in mammals, DHQS is a potential target for new antimicrobial agents, anti-parasitic agents and herbicides. The crystal structure of Helicobacter pylori DHQS (HpDHQS) complexed with NAD has been determined at 2.4-A resolution and was found to possess an N-terminal Rossmann-fold domain and a C-terminal alpha-helical domain. Structural comparison reveals that the binary complex adopts an open-state conformation and shares conserved residues in the binding pocket. Virtual docking of compounds into the active site of the HpDHQS structure using the GOLD docking program led to the identification of several inhibitors. The most active compound had an IC(50) value of 61 microM, which may serve as a lead for potent inhibitors.

Environmental signals that trigger bacterial pathogenesis and biofilm formation are mediated by changes in the level of cyclic dimeric guanosine monophosphate (c-di-GMP), a unique eubacterial second messenger. Tight regulation of cellular c-di-GMP concentration is governed by diguanylate cyclases and phosphodiesterases, which are responsible for its production and degradation, respectively. Here, we present the crystal structure of the diguanylate cyclase WspR, a conserved GGDEF domain-containing response regulator in Gram-negative bacteria, bound to c-di-GMP at an inhibitory site. Biochemical analyses revealed that feedback regulation involves the formation of at least three distinct oligomeric states. By switching from an active to a product-inhibited dimer via a tetrameric assembly, WspR utilizes a novel mechanism for modulation of its activity through oligomerization. Moreover, our data suggest that these enzymes can be activated by phosphodiesterases. Thus, in addition to the canonical pathways via phosphorylation of the regulatory domains, both product and enzyme concentration contribute to the coordination of c-di-GMP signaling. A structural comparison reveals resemblance of the oligomeric states to assemblies of GAF domains, widely used regulatory domains in signaling molecules conserved from archaea to mammals, suggesting a similar mechanism of regulation.

The increase in incidence of infectious diseases worldwide, particularly in developing countries, is worrying. Each year, 14 million people are killed by infectious diseases, mainly HIV/AIDS, respiratory infections, malaria and tuberculosis.. Despite the great burden in the poor countries, drug discovery to treat tropical diseases has come to a standstill. There is no interest by the pharmaceutical industry in drug development against the major diseases of the poor countries, since the financial return cannot be guaranteed. This has created an urgent need for new therapeutics to neglected diseases. A possible approach has been the exploitation of the inhibition of unique targets, vital to the pathogen such as the shikimate pathway enzymes, which are present in bacteria, fungi and apicomplexan parasites but are absent in mammals. The chorismate synthase (CS) catalyses the seventh step in this pathway, the conversion of 5-enolpyruvylshikimate-3-phosphate to chorismate. The strict requirement for a reduced flavin mononucleotide and the anti 1,4 elimination are both unusual aspects which make CS reaction unique among flavin-dependent enzymes, representing an important target for the chemotherapeutic agents development. In this review we present the main biochemical features of CS from bacterial and fungal sources and their difference from the apicomplexan CS. The CS mechanisms proposed are discussed and compared with structural data. The CS structures of some organisms are compared and their distinct features analyzed. Some known CS inhibitors are presented and the main characteristics are discussed. The structural and kinetics data reviewed here can be useful for the design of inhibitors.

The nonmevalonate isoprenoid pathway is an established target for antiinfective drug development. This paper describes high-throughput methods for the screening of 2C-methyl-D-erythritol synthase (IspC protein), 4-diphosphocytidyl-2C-methyl-D-erythritol synthase (IspD protein), 4-diphosphocytidyl-2C-methyl-D-erythritol kinase (IspE protein), and 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF protein) against large compound libraries. The assays use up to three auxiliary enzymes. They are all monitored photometrically at 340 nm and are robust as documented by Z-factors of >or=0.86. 13C NMR assays designed for hit verification via direct detection of the primary reaction product are also described. Enzyme-assisted methods for the preparation, on a multigram scale, of isoprenoid biosynthesis intermediates required as substrates for these assays are reported. Notably, these methods enable the introduction of single or multiple 13C labels as required for NMR-monitored assays. The preparation of 4-diphosphosphocytidyl-2C-methyl-D-erythritol 2-phosphate in multigram quantities is described for the first time.

Rat liver FAD-AMP lyase or FMN cyclase is the only known enzymatic source of the unusual flavin nucleotide riboflavin 4',5'-cyclic phosphate. To determine its molecular identity, a peptide-mass fingerprint of the purified rat enzyme was obtained. It pointed to highly related, mammalian hypothetical proteins putatively classified as dihydroxyacetone (Dha) kinases due to weaker homologies to biochemically proven Dha kinases of plants, yeasts, and bacteria. The human protein LOC26007 cDNA was used to design PCR primers. The product amplified from human brain cDNA was cloned, sequenced (GenBank Accession No. ), and found to differ from protein LOC26007 cDNA by three SNPs. Its heterologous expression yielded a protein active both as FMN cyclase and ATP-dependent Dha kinase, each activity being inhibited by the substrate(s) of the other. Cyclase and kinase activities copurified from rat liver extracts. Evidence supports that a single protein sustains both activities, probably in a single active center. Putative Dha kinases from other mammals are likely to be FMN cyclases too. Future work will profit from the availability of the structure of Citrobacter freundii Dha kinase, which contains substrate-interacting residues conserved in human Dha kinase/FMN cyclase.

Survival of bloodstream form Trypanosoma brucei, the agent of African sleeping sickness, normally requires mitochondrial gene expression, despite the absence of oxidative phosphorylation in this stage of the parasite's life cycle. Here we report that silencing expression of the alpha subunit of the mitochondrial F(1)-ATP synthase complex is lethal for bloodstream stage T. brucei as well as for T. evansi, a closely related species that lacks mitochondrial protein coding genes (i.e. is dyskinetoplastic). Our results suggest that the lethal effect is due to collapse of the mitochondrial membrane potential, which is required for mitochondrial function and biogenesis. We also identified a mutation in the gamma subunit of F(1) that is likely to be involved in circumventing the requirement for mitochondrial gene expression in another dyskinetoplastic form. Our data reveal that the mitochondrial ATP synthase complex functions in the bloodstream stage opposite to that in the insect stage and in most other eukaryotes, namely using ATP hydrolysis to generate the mitochondrial membrane potential.

DNA polymerase beta (beta-pol) plays a central role in repair of damaged DNA bases by base excision repair (BER) pathways. A predominant phenotype of beta-pol null mouse fibroblasts is hypersensitivity to the DNA-methylating agent methyl methanesulfonate. Residues in the 8-kDa domain of beta-pol that seem to interact with a known natural product beta-pol inhibitor, koetjapic acid, were identified by NMR chemical shift mapping. The data implicate the binding pocket as the hydrophobic cleft between helix-2 and helix-4, which provides the DNA binding and deoxyribose phosphate lyase activities of the enzyme. Nine structurally related synthetic compounds, containing aromatic or other hydrophobic groups in combination with two carboxylate groups, were then tested. They were found to bind to the same or a very similar region on the surface of the enzyme. The ability of these compounds to potentiate methyl methanesulfonate cytotoxicity, an indicator of cellular BER capacity, in wild-type and beta-pol null mouse fibroblasts, was next ascertained. The most active and beta-pol-specific of these agents, pamoic acid, was further characterized and found to be an inhibitor of the deoxyribose phosphate lyase and DNA polymerase activities of purified beta-pol on a BER substrate. Our results illustrate that NMR-based mapping techniques can be used in the design of small molecule enzyme inhibitors including those with potential use in a clinical setting.

A series of 2(Z)-2-benzylidene-6,7-dihydroxybenzofuran-3[2H]-ones was identified as potent inhibitors of bacterial chorismate synthase. The 2'-hydroxy-4'-pentoxy analogue 33 is a potent inhibitor of Streptococcus pneumoniae chorismate synthase.

The role of the active site metal in determining binding to 3-dehydroquinate synthase has been examined. Protocatechuic acid, catechol, and derivatives of these aromatics were synthesized that shared the common element of an ortho dihydroxylated benzene ring. Inhibition constants were determined for each aromatic as well as the variation of this inhibition as a function of whether Co(+2) or Zn(+2) was the active site metal ion.

Chorismate synthase catalyzes the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) to chorismate. The strict requirement for a reduced FMN cofactor and a trans-1,4-elimination are unusual. (6R)-6-Fluoro-EPSP was shown to be converted to chorismate stoichiometrically with enzyme-active sites in the presence of dithionite. This conversion was associated with the oxidation of FMN to give a stable flavin semiquinone. The IC(50) of the fluorinated substrate analogue was 0.5 and 250 microm with the Escherichia coli enzyme, depending on whether it was preincubated with the enzyme or not. The lack of dissociation of the flavin semiquinone and chorismate from the enzyme appears to be the basis of the essentially irreversible inhibition by this analogue. A dithionite-dependent transient formation of flavin semiquinone during turnover of (6S)-6-fluoro-EPSP has been observed. These reactions are best rationalized by radical chemistry that is strongly supportive of a radical mechanism occurring during normal turnover. The lack of activity with 5-deaza-FMN provides additional evidence for the role of flavin in catalysis by the E. coli enzyme.

A specific and highly potent inhibitor of diguanylate cyclase, the key regulatory enzyme of the cellulose synthesizing apparatus in the bacterium Acetobacter xylinum, was isolated from extracts of etiolated pea shoots (Pisum sativum). The inhibitor has been purified by a multistep procedure, and sufficient amounts of highly purified compound (3-8 mg) for spectral analysis were obtained. The structure of this compound was established as 3-O-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-galactopyranosyl-(1--> 2)-beta-D-glucuronopyranosyl soyasapogenol B 22-O-alpha-D-glucopyranoside. The structure was elucidated on the basis of susceptibility to various enzymes, chemical and spectral methods, such as GC-MS, FAB-MS, and the following types of 2D-NMR: COSY, ROESY, TOCSEY, HMQC, HMBC analyses. An identical or a very similar compound with identical biological activity was also isolated from A. xylinum, strongly suggesting that at least certain aspects of cellulose synthesis in the bacteria and in higher plants may be regulated in a similar manner. The content of this saponin in etiolated plants was about 0.04 mumol (g fresh tissue)-1.