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Botanical dietary supplements are widely used, but issues of authenticity, consistency, safety, and efficacy that complicate their poorly understood mechanism of action have prompted questions and concerns in the popular and scientific literature. Black cohosh ( Actaea racemosa L., syn. Cimicifuga racemosa , Nutt., Ranunculaceae) is a multicomponent botanical therapeutic used as a popular remedy for menopause and dysmenorrhea and explored as a treatment in breast and prostate cancer. However, its use and safety are controversial. A. racemosa tissues contain the bioactive serotonin analog N -methylserotonin, which is thought to contribute to the serotonergic activities of black cohosh-containing preparations. A. racemosa has several TDC -like genes hypothesized to encode tryptophan decarboxylases (TDCs) converting L-tryptophan to tryptamine, a direct serotonin precursor in plants. Expression of black cohosh TDC1 , TDC2 , and TDC3 in Saccharomyces cerevisiae resulted in the production of tryptamine. TDC1 and TDC3 had approximately fourfold higher activity than TDC2, which was attributable to a variable Cys/Ser active site residue identified by site-directed mutagenesis. Co-expression in yeast of the high-activity black cohosh TDCs with the next enzyme in serotonin biosynthesis, tryptamine 5-hydroxylase (T5H), from rice ( Oryza sativa ) resulted in the production of serotonin, whereas co-expression with low-activity TDCs did not, suggesting that TDC activity is a rate-limiting step in serotonin biosynthesis. Two T5H-like sequences were identified in A. racemose, but their co-expression with the high-activity TDCs in yeast did not result in serotonin production. TDC expression was detected in several black cohosh tissues, and phytochemical analysis using LC-MS revealed several new tryptamines, including tryptamine and serotonin, along with N -methylserotonin and, interestingly, N - N -dimethyl-5-hydroxytryptamine (bufotenine), which may contribute to hepatotoxicity. Incubation of A. racemosa leaves with tryptamine and N -methyltryptamine resulted in increased concentrations of serotonin and N -methylserotonin, respectively, suggesting that methylation of tryptamine precedes hydroxylation in the biosynthesis of N -methylserotonin. This work indicates a significantly greater variety of serotonin derivatives in A. racemosa than previously reported. Moreover, the activities of the TDCs underscore their key role in the production of serotonergic compounds in A. racemosa . Finally, it is proposed that tryptamine is first methylated and then hydroxylated to form the black cohosh signature compound N -methylserotonin.

Recently, we showed that the fused chorismate-utilizing enzyme from the antibiotic-producing soil bacterium Streptomyces venezuelae is an anthranilate synthase (designated SvAS), not a 2-amino-2-deoxyisochorismate (ADIC) synthase, as was predicted based on its amino acid sequence similarity to the phenazine biosynthetic enzyme PhzE (an ADIC synthase). Here, we report the characterization of SvAS using steady-state kinetics, gel filtration chromatography, and laser light scattering. The recombinant His-tagged enzyme has Michaelis constants Km with respect to substrates chorismate and glutamine of 8.2 ± 0.2 μM and 0.84 ± 0.05 mM, respectively, and a catalytic rate constant k cat of 0.57 ± 0.02 s(-1) at 30 °C. Unlike most other anthranilate synthases, SvAS does not utilize ammonia as a substrate. The enzyme is competitively but non-cooperatively inhibited by tryptophan (K i = 11.1 ± 0.1 μM) and is active as a monomer. The finding that SvAS is a monomer jibes with the variety of association modes that have been observed for anthranilate synthases from different microorganisms, and it identifies the enzyme's minimal functional unit as a single TrpE-TrpG pair.

The phenazines are a class of over 150 nitrogen-containing aromatic compounds of bacterial and archeal origin. Their redox properties not only explain their activity as broad-specificity antibiotics and virulence factors but also enable them to function as respiratory pigments, thus extending their importance to the primary metabolism of phenazine-producing species. Despite their discovery in the mid-19th century, the molecular mechanisms behind their biosynthesis have only been unraveled in the last decade. Here, we review the contribution of structural biology that has led to our current understanding of phenazine biosynthesis., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The diversity of useful compounds produced by plant secondary metabolism has stimulated broad systems biology approaches to identify the genes involved in their biosynthesis. Systems biology studies in non-model plants pose interesting but addressable challenges, and have been greatly facilitated by the ability to grow and maintain plants, develop laboratory culture systems, and profile key metabolites in order to identify critical genes involved their biosynthesis. In this chapter we describe a suite of approaches that have been useful in Actaea racemosa (L.; syn. Cimicifuga racemosa, Nutt., black coshosh), a non-model medicinal plant with no genome sequence and little horticultural information available, that have led to the development of initial gene-metabolite relationships for the production of several bioactive metabolites in this multicomponent botanical therapeutic, and that can be readily applied to a wide variety of under-characterized medicinal plants.

The structure of PA5508 from Pseudomonas aeruginosa, a glutamine synthetase (GS) homologue, has been determined at 2.5 Å. Surprisingly, PA5508 forms single hexameric rings rather than the stacked double rings that are characteristic of GS. The C-terminal helical thong motif that links GS rings is present in PA5508; however, it is folded back toward the core of its own polypeptide, preventing it from interacting with a second ring. Interestingly, PA5508 displays a clear preference for aromatic amine substrates. Unique aspects of the structure illustrate how the enzyme is able to catalyze reactions involving bulky amines rather than ammonia.

PabB, aminodeoxychorismate synthase, is the chorismic acid binding component of the heterodimeric PabA-PabB complex that converts chorismic acid to 4-amino-4-deoxychorismate, a precursor of p-aminobenzoate and folic acid in microorganisms. The second component, a glutamine amidotransferase subunit, PabA, generates ammonia that is channeled to the PabB active site where it attacks C4 of a chorismate-derived intermediate that is covalently bound, through C2, to an active site lysine residue. The presence of a PIKGT motif was, until recently, believed to allow discrimination of PabB enzymes from the closely related enzyme anthranilate synthase, which typically contains a PIAGT active site motif and does not form a covalent enzyme-substrate intermediate with chorismate. A subclass of PabB enzymes that employ an alternative mechanism requiring 2 equiv of ammonia from glutamine and that feature a noncovalently bound 2-amino-2-deoxyisochorismate intermediate was recently identified. Here we report the 2.25 Å crystal structure of PabB from the emerging pathogen Stenotrophomonas maltophilia. It is the first reported structure of a PabB that features the PIAGT motif. Surprisingly, no dedicated pabA is evident in the genome of S. maltophilia, suggesting that another cellular amidotransferase is able to fulfill the role of PabA in this organism. Evaluation of the ammonia-dependent aminodeoxychorismate synthase activity of S. maltophilia PabB alone revealed that it is virtually inactive. However, in the presence of a heterologous PabA surrogate, typical levels of activity were observed using either glutamine or ammonia as the nitrogen source. Additionally, the structure suggests that a key segment of the polypeptide can remodel itself to interact with a nonspecialized or shared amidotransferase partner in vivo. The structure and mass spectral analysis further suggest that S. maltophilia PabB, like Escherichia coli PabB, binds tryptophan in a vestigial regulatory site. The observation that the binding site is unoccupied in the crystal structure, however, suggests the affinity may be low relative to that of E. coli PabB.

To date, lacking of a clinically-suitable source of engraftable human stem/progenitor cells with adequate neurogenic potential has been the major setback in developing effective cell-based therapies against a wide range of neurological disorders. Derivation of human embryonic stem cells (hESCs) provides a powerful tool to investigate the molecular controls in human embryonic neurogenesis as well as an unlimited source to generate the diversity of human neuronal cell types in the developing CNS for repair. However, realizing the developmental and therapeutic potential of hESCs has been hindered by conventional multi-lineage differentiation of pluripotent cells, which is uncontrollable, inefficient, highly variable, difficult to reproduce and scale-up. We recently identified retinoic acid (RA) as sufficient to induce the specification of neuroectoderm direct from the pluripotent state of hESCs under defined platform and trigger progression to human neuronal progenitors (hESC-I hNuPs) and neurons (hESC-I hNus) in the developing CNS with high efficiency, which enables hESC neuronal lineage-specific differentiation and opens the door to investigate human embryonic neurogenesis using the hESC model system. In this study, genome-scale profiling of microRNA (miRNA) differential expression patterns in hESC neuronal lineage-specific progression was used to identify molecular signatures of human embryonic neurogenesis. These in vitro neuroectoderm-derived human neuronal cells have acquired a neuron al identity by down-regulating pluripotence-associated miRNAs and inducing the expression of miRNAs linked to regulating human CNS development to high levels in a stage-specific manner, including silencing of the prominent pluripotence-associated hsa-miR-302 family and drastic expression increases of the Hox hsa-miR-10 and let-7 miRNAs. Following transplantation, hESC-I hNuPs engrafted and yielded well-integrated neurons at a high prevalence within neurogenic regions of the brain. In 3D culture, these hESC-I hNuPs proceeded to express subtype neuronal markers, such as dopaminergic and motor neurons, demonstrating their therapeutic potential for CNS repair. Our study provides critical insight into molecular neurogenesis in human embryonic development as well as offers an adequate human neurogenic cell source in high purity and large quantity for scale-up CNS regeneration.

We introduce the wetting barrier ratchet, a digital microfluidic technology for directed drop transport in an open air environment. Cyclic drop footprint oscillations initiated by orthogonal vibrations as low as 37 μm in amplitude at 82 Hz are rectified into fast (mm/s) and controlled transport along a fabricated ratchet design. The ratchet is made from a simple wettability pattern atop a microscopically flat surface consisting of periodic semi-circular hydrophilic features on a hydrophobic background. The microfluidic ratchet capitalizes on the asymmetric contact angle hysteresis induced by the curved features to drive transport. In comparison to the previously reported texture ratchets, wetting barrier ratchets require 3-fold lower actuation amplitudes for a 10 μL drop, have a simplified fabrication, and can be made optically flat for applications where transparency is paramount.

Protein-protein interactions identified through high-throughput proteomics efforts continue to advance our understanding of the protein interactome. In addition to highly specific protein-protein interactions, it is becoming increasingly more common for yeast two-hybrid, pull-down assays, and other proteomics techniques to identify multiple protein ligands that bind to the same target protein. A resulting challenge is to accurately characterize the assembly of these multiprotein complexes and the competition among multiple protein ligands for a given target. The Association of Biomolecular Resource Facilities-Molecular Interactions Research Group recently conducted a benchmark study to assess participants' ability to correctly describe the interactions between two protein ligands and their target protein using primarily biosensor technologies, such as surface plasmon resonance. Participants were provided with microgram quantities of three proteins (A, B, and C) and asked to determine if a ternary A-B-C complex can form or if protein-B and protein-C bind competitively to protein-A. This article will summarize the experimental approaches taken by participants to characterize the molecular interactions, the interpretation of the data, and the results obtained using different biosensor instruments.

To date, lacking of a clinically-suitable human cardiac cell source with adequate myocardium regenerative potential has been the major setback in regenerating the damaged human myocardium. Pluripotent Human Embryonic Stem Cells (hESCs) proffer unique revenue to generate a large supply of cardiac lineage-committed cells as human myocardial grafts for cell-based therapy. Due to the prevalence of heart disease worldwide and acute shortage of donor organs or human myocardial grafts, there is intense interest in developing hESC-based therapy for heart disease and failure. However, realizing the potential of hESCs has been hindered by the inefficiency and instability of generating cardiac cells from pluripotent cells through uncontrollable multi-lineage differentiation. In addition, the need for foreign biologics for derivation, maintenance, and differentiation of hESCs may make direct use of such cells and their derivatives in patients problematic. Understanding the requirements for sustaining pluripotentce and self-renewal of hESCs will provide the foundation for de novo derivation and long-term maintenance of biologics-free hESCs under optimal yet well-defined culture conditions from which they can be efficiently directed towards clinically-relevant lineages for therapies. We previously reported the resolving of the elements of a defined culture system, serving as a platform for effectively directing pluripotent hESCs uniformly towards a cardiac lineage-specific fate by small molecule induction. In this study, we found that, under the defined culture conditions, primitive endoderm-like (PEL) cells constitutively emerged and acted through the activin-A-SMAD pathway in a paracrine fashion to sustain the epiblast pluripotence of hESCs. Such defined conditions enable the spontaneous unfolding of inherent early embryogenesis processes that, in turn, aid efficient clonal propagation and de novo derivation of stable biologics-free hESCs from blastocysts that can be directly differentiated into a large supply of clinically-suitable human myocardial grafts across the spectrum of developmental stages using small molecule induction for cardiovascular repair.

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging(1-3). Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells(1-3). Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration(1,4-7). However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity(7-10). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic(11-13). To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules(14) (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders(1, 14). And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons(1, 14, 15). However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

The structure of EhpF, a 41 kDa protein that functions in the biosynthetic pathway leading to the broad-spectrum antimicrobial compound D-alanylgriseoluteic acid (AGA), is reported. A cluster of approximately 16 genes, including ehpF, located on a 200 kbp plasmid native to certain strains of Pantoea agglomerans encodes the proteins that are required for the conversion of chorismic acid to AGA. Phenazine-1,6-dicarboxylate has been identified as an intermediate in AGA biosynthesis and deletion of ehpF results in accumulation of this compound in vivo. The crystallographic data presented here reveal that EhpF is an atypical member of the acyl-CoA synthase or ANL superfamily of adenylating enzymes. These enzymes typically catalyze two-step reactions involving adenylation of a carboxylate substrate followed by transfer of the substrate from AMP to coenzyme A or another phosphopantetheine. EhpF is distinguished by the absence of the C-terminal domain that is characteristic of enzymes from this family and is involved in phosphopantetheine binding and in the second half of the canonical two-step reaction that is typically observed. Based on the structure of EhpF and a bioinformatic analysis, it is proposed that EhpF and EhpG convert phenazine-1,6-dicarboxylate to 6-formylphenazine-1-carboxylate via an adenylyl intermediate.

Pseudomonas quinolone signal (PQS), 2-heptyl-3-hydroxy-4-quinolone, is an intercellular alkyl quinolone signaling molecule produced by the opportunistic pathogen Pseudomonas aeruginosa. Alkyl quinolone signaling is an atypical system that, in P. aeruginosa, controls the expression of numerous virulence factors. PQS is synthesized from the tryptophan pathway intermediate, anthranilate, which is derived either from the kynurenine pathway or from an alkyl quinolone specific anthranilate synthase encoded by phnAB. Anthranilate is converted to PQS by the enzymes encoded by the pqsABCDE operon and pqsH. PqsA forms an activated anthraniloyl-CoA thioester that shuttles anthranilate to the PqsD active site where it is transferred to Cys112 of PqsD. In the only biochemically characterized reaction, a condensation then occurs between anthraniloyl-PqsD and malonyl-CoA or malonyl-ACP, a second PqsD substrate, forming 2,4-dihydroxyquinoline (DHQ). The role PqsD plays in the biosynthesis of other alkyl quinolones, such as PQS, is unclear, though it has been reported to be required for their production. No evidence exists that DHQ is a PQS precursor, however. Here we present a structural and biophysical characterization of PqsD that includes several crystal structures of the enzyme, including that of the PqsD-anthranilate covalent intermediate and the inactive Cys112Ala active site mutant in complex with anthranilate. The structure reveals that PqsD is structurally similar to the FabH and chalcone synthase families of fatty acid and polyketide synthases. The crystallographic asymmetric unit contains a PqsD dimer. The PqsD monomer is composed of two nearly identical approximately 170-residue alphabetaalphabetaalpha domains. The structures show anthranilate-liganded Cys112 is positioned deep in the protein interior at the bottom of an approximately 15 A long channel while a second anthraniloyl-CoA molecule is waiting in the cleft leading to the protein surface. Cys112, His257, and Asn287 form the FabH-like catalytic triad of PqsD. The C112A mutant is inactive, although it still reversibly binds anthraniloyl-CoA. The covalent complex between anthranilate and Cys112 clearly illuminates the orientation of key elements of the PqsD catalytic machinery and represents a snapshot of a key point in the catalytic cycle.

The Arabidopsis thaliana resistance gene RPW8 triggers the hypersensitive response (HR) to restrict powdery mildew infection via the salicylic acid-dependent signaling pathway. To further understand how RPW8 signaling is regulated, we have conducted a genetic screen to identify mutations enhancing RPW8-mediated HR-like cell death (designated erh). Here, we report the isolation and characterization of the Arabidopsis erh1 mutant, in which the At2g37940 locus is knocked out by a T-DNA insertion. Loss of function of ERH1 results in salicylic acid accumulation, enhanced transcription of RPW8 and RPW8-dependent spontaneous HR-like cell death in leaf tissues, and reduction in plant stature. Sequence analysis suggests that ERH1 may encode the long-sought Arabidopsis functional homolog of yeast and protozoan inositolphosphorylceramide synthase (IPCS), which converts ceramide to inositolphosphorylceramide. Indeed, ERH1 is able to rescue the yeast aur1 mutant, which lacks the IPCS, and the erh1 mutant plants display reduced ( approximately 53% of wild type) levels of leaf IPCS activity, indicating that ERH1 encodes a plant IPCS. Consistent with its biochemical function, the erh1 mutation causes ceramide accumulation in plants expressing RPW8. These data reinforce the concept that sphingolipid metabolism (specifically, ceramide accumulation) plays an important role in modulating plant programmed cell death associated with defense.

The human pathogen Pseudomonas aeruginosa produces pyocyanin, a blue-pigmented phenazine derivative, which is known to play a role in virulence. Pyocyanin is produced from chorismic acid via the phenazine pathway, nine proteins encoded by a gene cluster. Phenazine-1-carboxylic acid, the initial phenazine formed, is converted to pyocyanin in two steps that are catalyzed by the enzymes PhzM and PhzS. PhzM is an adenosylmethionine dependent methyltransferase, and PhzS is a flavin dependent hydroxylase. It has been shown that PhzM is only active in the physical presence of PhzS, suggesting that a protein-protein interaction is involved in pyocyanin formation. Such a complex would prevent the release of 5-methyl-phenazine-1-carboxylate, the putative intermediate, and an apparently unstable compound. Here, we describe the three-dimensional structure of PhzS, solved by single anomalous dispersion, at a resolution of 2.4 A. The structure reveals that PhzS is a member of the family of aromatic hydroxylases characterized by p-hydroxybenzoate hydroxylase. The flavin cofactor of PhzS is in the solvent exposed out orientation typically seen in unliganded aromatic hydroxylases. The PhzS flavin, however, appears to be held in a strained conformation by a combination of stacking interactions and hydrogen bonds. The structure suggests that access to the active site is gained via a tunnel on the opposite side of the protein from where the flavin is exposed. The C-terminal 23 residues are disordered as no electron density is present for these atoms. The probable location of the C-terminus, near the substrate access tunnel, suggests that it may be involved in substrate binding as has been shown for another structural homologue, RebC. This region also may be an element of a PhzM-PhzS interface. Aromatic hydroxylases have been shown to catalyze electrophilic substitution reactions on activated substrates. The putative PhzS substrate, however, is electron deficient and unlikely to act as a nucleophile, suggesting that PhzS may use a different mechanism than its structural relatives.

The electron carrier menaquinone is one of many important bacterial metabolites that are derived from the key intermediate chorismic acid. MenF, the first enzyme in the menaquinone pathway, catalyzes the isomerization of chorismate to isochorismate. Here, an improved structure of MenF in a new crystal form is presented. The structure, solved at 2.0 angstroms resolution in complex with magnesium, reveals a well defined closed active site. Existing evidence suggests that the mechanism of the reaction catalyzed by MenF involves nucleophilic attack of a water molecule on the chorismate ring. The structure reveals a well defined water molecule located in an appropriate position for activation by Lys190 and attack on the substrate.

Pyocyanin is a biologically active phenazine produced by the human pathogen Pseudomonas aeruginosa. It is thought to endow P. aeruginosa with a competitive growth advantage in colonized tissue and is also thought to be a virulence factor in diseases such as cystic fibrosis and AIDS where patients are commonly infected by pathogenic Pseudomonads due to their immunocompromised state. Pyocyanin is also a chemically interesting compound due to its unusual oxidation-reduction activity. Phenazine-1-carboxylic acid, the precursor to the bioactive phenazines, is synthesized from chorismic acid by enzymes encoded in a seven-gene cistron in P. aeruginosa and in other Pseudomonads. Phenzine-1-carboxylic acid is believed to be converted to pyocyanin by the sequential actions of the putative S-adenosylmethionine-dependent N-methyltransferase PhzM and the putative flavin-dependent hydroxylase PhzS. Here we report the 1.8 A crystal structure of PhzM determined by single anomalous dispersion. Unlike many methyltransferases, PhzM is a dimer in solution. The 36 kDa PhzM polypeptide folds into three domains. The C-terminal domain exhibits the alpha/beta-hydrolase fold typical of small molecule methyltransferases. Two smaller N-terminal domains form much of the dimer interface. Structural alignments with known methyltransferases show that PhzM is most similar to the plant O-methyltransferases that are characterized by an unusual intertwined dimer interface. The structure of PhzM contains no ligands, and the active site is open and solvent-exposed when compared to structures of similar enzymes. In vitro experiments using purified PhzM alone demonstrate that it has little or no ability to methylate phenzine-1-carboxylic acid. However, when the putative hydroxylase PhzS is included, pyocyanin is readily produced. This observation suggests that a mechanism has evolved in P. aeruginosa that ensures efficient production of pyocyanin via the prevention of the formation and release of an unstable and potentially deleterious intermediate.

Bacteria have evolved elaborate schemes that help them thrive in environments where free iron is severely limited. Siderophores such as yersiniabactin are small iron-scavenging molecules that are deployed by bacteria during iron starvation. Several studies have linked siderophore production and virulence. Yersiniabactin, produced by several Enterobacteriaceae, is derived from the key metabolic intermediate chorismic acid via its conversion to salicylate by salicylate synthase. Crystals of salicylate synthase from the uropathogen Escherichia coli CFT073 have been grown by vapour diffusion using polyethylene glycol as the precipitant. The monoclinic (P2(1)) crystals diffract to 2.5 A. The unit-cell parameters are a = 57.27, b = 164.07, c = 59.04 A, beta = 108.8 degrees. The solvent content of the crystals is 54% and there are two molecules of the 434-amino-acid protein in the asymmetric unit. It is anticipated that the structure will reveal key details about the reaction mechanism and the evolution of salicylate synthase.

Canonical glutathione (GSH) transferases are dimeric proteins with subunits composed of an N-terminal GSH binding region (domain 1) and a C-terminal helical region (domain 2). The stabilities of several GSH transferase dimers are dependent upon two groups of interactions between domains 1 and 2 of opposing subunits: a hydrophobic ball-and-socket motif and a buried charge cluster motif. In rGSTM1-1, these motifs involve residues F56 and R81, respectively. The structural basis for the effects of mutating F56 to different residues on dimer stability and function has been reported (Codreanu et al. (2005) Biochemistry 44, 10605-10612). Here, we show that the simultaneous disruption of both motifs in the F56S/R81A mutant causes complete dissociation of the dimer to a monomeric protein on the basis of gel filtration chromatography and multiple-angle laser light scattering. The fluorescence and far-UV CD properties of the double mutant as well as the kinetics of amide H/D exchange along the polypeptide backbone suggest that the monomer has a globular structure that is similar to a single subunit in the native protein. However, the mutant monomer has severely impaired catalytic activity, suggesting that the dimer interface is vital for efficient catalysis. Backbone amide H/D exchange kinetics in the F56S and F56S/R81A mutants indicate that a reorganization of the loop structure between helix alpha2 and strand beta3 near the active site is responsible for the decreased catalytic activity of the monomer. In addition, the junction between the alpha4 and alpha5 helices in F56S/R81R shows decreased H/D exchange, indicating another structural change that may affect catalysis. Although the native subunit interface is important for dimer stability, urea-induced unfolding of the F56S/R81A mutant suggests that the interface is not essential for the thermodynamic stability of individual subunits. The H/D exchange data reveal a possible molecular basis for the folding cooperativity observed between domains 1 and 2.

The solution structure of the 154-residue conserved hypothetical protein HI0004 has been determined using multidimensional heteronuclear NMR spectroscopy. HI0004 has sequence homologs in many organisms ranging from bacteria to humans and is believed to be essential in Haemophilus influenzae, although an exact function has yet to be defined. It has a alpha-beta-alpha sandwich architecture consisting of a central four-stranded beta-sheet with the alpha2-helix packed against one side of the beta-sheet and four alpha-helices (alpha1, alpha3, alpha4, alpha5) on the other side. There is structural homology with the eukaryotic matrix metalloproteases (MMPs), but little sequence similarity except for a conserved region containing three histidines that appears in both the MMPs and throughout the HI0004 family of proteins. The solution structure of HI0004 is compared with the X-ray structure of an Aquifex aeolicus homolog, AQ_1354, which has 36% sequence identity over 148 residues. Despite this level of sequence homology, significant differences exist between the two structures. These differences are described along with possible functional implications of the structures.

PhzG is a flavin-dependent oxidase that is believed to play a role in phenazine antibiotic synthesis in various bacteria, including Pseudomonas. Phenazines are chorismic acid derivatives that provide the producing organisms, including the opportunistic pathogen P. aeruginosa, with a competitive growth advantage. Here, the crystal structures of PhzG from both P. aeruginosa and P. fluorescens solved in an unliganded state at 1.9 and 1.8 A resolution, respectively, are described. Although the specific reaction in phenazine biosynthesis catalyzed by PhzG is unknown, the structural data indicates that PhzG is closely related to pyridoxine-5'-phosphate oxidase, the Escherichia coli pdxH gene product, which catalyzes the final step in pyridoxal-5'-phosphate (PLP) biosynthesis. A previous proposal suggested that the physiological substrate of PhzG to be 2,3-dihydro-3-hydroxyanthranilic acid (DHHA), a phenazine precursor produced by the sequential actions of the PhzE and PhzD enzymes on chorismate, and that two DHHA molecules dimerized in another enzyme-catalyzed reaction to yield phenazine-1-carboxylate. However, it was not possible to demonstrate any in vitro activity upon incubation of PhzG and DHHA. Interestingly, analysis of the in vitro activities of PhzG in combination with PhzF suggests that PhzF acts on DHHA and that PhzG then reacts with a non-aromatic tricyclic phenazine precusor to catalyze an oxidation/aromatization reaction that yields phenazine-1-carboxylate. It is proposed that phzG arose by duplication of pdxH and that the subtle differences seen between the structures of PhzG and PdxH correlate with the loss of the ability of PhzG to catalyze PLP formation. Sequence alignments and superimpositions of the active sites of PhzG and PdxH reveal that the residues that form a positively charged pocket around the phosphate of PLP in the PdxH-PLP complex are not conserved in PhzG, consistent with the inability of phosphorylated compounds to serve as substrates for PhzG.

Phenazines, including pyocyanin and iodonin, are biologically active compounds that are believed to confer producing organisms with a competitive growth advantage, and also are thought to be virulence factors in certain diseases including cystic fibrosis. The basic, tricyclic phenazine ring system is synthesized in a series of poorly characterized steps by enzymes encoded in a seven-gene cistron in Pseudomonas and other organisms. Despite the biological importance of these compounds, and our understanding of their mode of action, the biochemistry and mechanisms of phenazine biosynthesis are not well resolved. Here we report the 1.8 A crystal structure of PhzF, a key enzyme in phenazine biosynthesis, solved by molecular replacement. PhzF is structurally similar to the lysine biosynthetic enzyme diaminopimelate epimerase, sharing an unusual fold consisting of two nearly identical domains with the active site located in an occluded cleft between the domains. Unlike diaminopimelate epimerase, PhzF is a dimer in solution. The two apparently independent active sites open toward opposite sides of the dimer and are occupied by sulfate ions in the structure. In vitro experiments using a mixture of purified PhzF, -A, -B, and -G confirm that phenazine-1-carboxylic acid (PCA) is readily produced from trans-2,3-dihydro-3-hydroxyanthranilic acid (DHHA) without aid of other cellular factors. PhzA, -B, and -G have no activity toward DHHA. However, in the presence of PhzF, individually or in combinations, they accelerate the formation of PCA from DHHA and therefore appear to function after the action of PhzF. Surprisingly, PhzF is itself capable of producing PCA, albeit slowly, from DHHA. These observations suggest that PhzF catalyzes the initial step in the conversion of DHHA to PCA, probably via a rearrangement reaction yielding the more reactive 3-oxo analogue of DHHA, and that subsequent steps can occur spontaneously. A hypothetical model for how DHHA binds to the PhzF active site suggests that Glu45 and Asp208 could act as general acid-base catalysts in a rearrangement reaction. Given that four reactions lie between DHHA and PCA, ketone formation, ring formation, decarboxylation, and oxidation, we hypothesize that the similar PhzA and -B proteins catalyze ring formation and thus may be more than noncatalytic accessory proteins. PhzG is almost certainly an oxidase and is predicted to catalyze the final oxidation/aromatization reaction.

The class kappa glutathione (GSH) transferase is an enzyme that resides in the mitochondrial matrix. Its relationship to members of the canonical GSH transferase superfamily has remained an enigma. The three-dimensional structure of the class kappa enzyme from rat (rGSTK1-1) in complex with GSH has been solved by single isomorphous replacement with anomalous scattering at a resolution of 2.5 A. The structure reveals that the enzyme is more closely related to the protein disulfide bond isomerase, dsbA, from Escherichia coli than it is to members of the canonical superfamily. The structures of rGSTK1-1 and the canonical superfamily members indicate that the proteins folds have diverged from a common thioredoxin/glutaredoxin progenitor but did so by different mechanisms. The mitochondrial enzyme, therefore, represents a fourth protein superfamily that supports GSH transferase activity. The thioredoxin domain functions in a manner that is similar to that seen in the canonical enzymes by providing key structural elements for the recognition of GSH. The hydroxyl group of S16 is within hydrogen-bonding distance of the sulfur of bound GSH and is, in part, responsible for the ionization of the thiol in the E*GSH complex (pKa = 6.4 +/- 0.1). Preequilibrium kinetic experiments indicate that the k(on) for GSH is 1 x 10(5) M(-1) s(-1) and k(off) for GS- is approximately 8 s(-1) and relatively slow with respect to turnover with 1-chloro-2, 4-dinitrobenzene (CDNB). As a result, the KM(GSH) (11 mM) is much larger than the apparent Kd(GSH) (90 microM). The active site has a relatively open access channel that is flanked by disordered loops that may explain the relatively high turnover number (280 s(-1) at pH 7.0) toward CDNB. The disordered loops form an extensive contiguous patch on one face of the dimeric enzyme, a fact that suggests that the protein surface may interact with a membrane or other protein partner.

Multiple sequence alignments of the eight glutathione (GSH) transferase homologues encoded in the genome of Escherichia coli were used to define a consensus sequence for the proteins. The consensus sequence was analyzed in the context of the three-dimensional structure of the gst gene product (EGST) obtained from two different crystal forms of the enzyme. The enzyme consists of two domains. The N-terminal region (domain I) has a thioredoxin-like alpha/beta-fold, while the C-terminal domain (domain II) is all alpha-helical. The majority of the consensus residues (12/17) reside in the N-terminal domain. Fifteen of the 17 residues are involved in hydrophobic core interactions, turns, or electrostatic interactions between the two domains. The results suggest that all of the homologues retain a well-defined group of structural elements both in and between the N-terminal alpha/beta domain and the C-terminal domain. The conservation of two key residues for the recognition motif for the gamma-glutamyl-portion of GSH indicates that the homologues may interact with GSH or GSH analogues such as glutathionylspermidine or alpha-amino acids. The genome context of two of the homologues forms the basis for a hypothesis that the b2989 and yibF gene products are involved in glutathionylspermidine and selenium biochemistry, respectively., (Copyright 2003 Wiley-Liss, Inc.)

PhzD from Pseudomonas aeruginosa is an isochorismatase involved in phenazine biosynthesis. Phenazines are antimicrobial compounds that provide Pseudomonas with a competitive advantage in certain environments and may be partly responsible for the persistence of Pseudomonas infections. In vivo, PhzD catalyzes the hydrolysis of the vinyl ether functional group of 2-amino-2-deoxyisochorismate, yielding pyruvate and trans-2,3-dihydro-3-hydroxyanthranilic acid, which is then utilized in the phenazine biosynthetic pathway. PhzD also catalyzes hydrolysis of the related vinyl ethers isochorismate, chorismate, and 4-amino-4-deoxychorismate. Here we report the 1.5 A crystal structure of native PhzD, and the 1.6 A structure of the inactive D38A variant in complex with isochorismate. The structures reveal that isochorismate binds to the PhzD active site in a trans-diaxial conformation, and superposition of the structures indicates that the methylene pyruvyl carbon of isochorismate is adjacent to the side chain carboxylate of aspartate 38. The proximity of aspartate 38 to isochorismate and the complete loss of activity resulting from the conversion of aspartate 38 to alanine suggest a mechanism in which the carboxylate acts as a general acid to protonate the substrate, yielding a carbocation/oxocarbonium ion that is then rapidly hydrated to form a hemiketal intermediate, which then decomposes spontaneously to products. The structure of PhzD is remarkably similar to other structures from a subfamily of alpha/beta-hydrolase enzymes that includes pyrazinamidase and N-carbamoylsarcosine amidohydrolase. However, PhzD catalyzes unrelated chemistry and lacks a nucleophilic cysteine found in its close structural relatives. The vinyl ether hydrolysis catalyzed by PhzD represents yet another example of the catalytic diversity seen in the alpha/beta-hydrolase family, whose members are also known to hydrolyze amides, phosphates, phosphonates, epoxides, and C-X bonds.

Microcalorimetry and high-performance liquid chromatography (HPLC) have been used to conduct a thermodynamic investigation of reactions catalyzed by PABA synthase, the enzyme located at the first step in the shikimic acid metabolic pathway leading from chorismate to 4-aminobenzoate (PABA). The overall biochemical reaction catalyzed by the PabB and PabC components of PABA synthase is: chorismate(aq)+ammonia(aq)=4-aminobenzoate(aq)+pyruvate(aq)+H(2)O(l). This reaction can be divided into two partial reactions involving the intermediate 4-amino-4-deoxychorismate (ADC): chorismate(aq)+ammonia(aq)=ADC(aq)+H(2)O(l) and ADC(aq)=4-aminobenzoate(aq)+pyruvate(aq). Microcalorimetric measurements were performed on all three of these reactions at a temperature of 298.15 K and pH values in the range 8.72-8.77. Equilibrium measurements were performed on the first partial (ADC synthase) reaction at T=298.15 K and at pH=8.78. The saturation molality of 4-aminobenzoate(cr) in water is (0.00382+/-0.0004) mol kg(-1) at T=298.15 K. The results of the equilibrium and calorimetric measurements were analyzed in terms of a chemical equilibrium model that accounts for the multiplicity of ionic states of the reactants and products. These calculations gave thermodynamic quantities at the temperature 298.15 K and an ionic strength of zero for chemical reference reactions involving specific ionic forms. For the reaction: chorismate(2-)(aq)+NH(4)(+)(aq)=ADC(-)(aq)+H(2)O(l), K=(10.8+/-4.2) and Delta(r)H(m)(o)=-(35+/-15) kJ mol(-1). For the reaction: ADC(-)(aq)=4-aminobenzoate(-)(aq)+pyruvate(-)(aq)+H(+)(aq), Delta(r)H(m)(o)=-(139+/-23) kJ mol(-1). For the reaction: chorismate(2-)(aq)+NH(4)(+)(aq)=4-aminobenzoate(-)(aq)+pyruvate(-)(aq)+H(2)O(l)+H(+)(aq), Delta(r)H(m)(o)=-(174+/-6) kJ mol(-1). Thermodynamic cycle calculations were used to calculate thermodynamic quantities for three additional reactions that utilize L-glutamine rather than ammonia and that are pertinent to this branch point of the shikimic acid pathway. The quantities obtained in this study permit the calculation of the position of equilibrium of these reactions as a function of temperature, pH, and ionic strength. Values of the apparent equilibrium constants and the standard transformed Gibbs energy changes Delta(r)G'(m)(o) under approximately physiological conditions are given.

The crystal structure of the YrbI protein from Haemophilus influenzae (HI1679) was determined at a 1.67-A resolution. The function of the protein had not been assigned previously, and it is annotated as hypothetical in sequence databases. The protein exhibits the alpha/beta-hydrolase fold (also termed the Rossmann fold) and resembles most closely the fold of the L-2-haloacid dehalogenase (HAD) superfamily. Following this observation, a detailed sequence analysis revealed remote homology to two members of the HAD superfamily, the P-domain of Ca(2+) ATPase and phosphoserine phosphatase. The 19-kDa chains of HI1679 form a tetramer both in solution and in the crystalline form. The four monomers are arranged in a ring such that four beta-hairpin loops, each inserted after the first beta-strand of the core alpha/beta-fold, form an eight-stranded barrel at the center of the assembly. Four active sites are located at the subunit interfaces. Each active site is occupied by a cobalt ion, a metal used for crystallization. The cobalt is octahedrally coordinated to two aspartate side-chains, a backbone oxygen, and three solvent molecules, indicating that the physiological metal may be magnesium. HI1679 hydrolyzes a number of phosphates, including 6-phosphogluconate and phosphotyrosine, suggesting that it functions as a phosphatase in vivo. The physiological substrate is yet to be identified; however the location of the gene on the yrb operon suggests involvement in sugar metabolism., (Copyright 2002 Wiley‐Liss, Inc.)

Aminodeoxychorismate synthase is part of a heterodimeric complex that catalyzes the two-step biosynthesis of 4-amino-4-deoxychorismate, a precursor of p-aminobenzoate and folate in microorganisms. In the first step, a glutamine amidotransferase encoded by the pabA gene generates ammonia as a substrate that, along with chorismate, is used in the second step, catalyzed by aminodeoxychorismate synthase, the product of the pabB gene. Here we report the X-ray crystal structure of Escherichia coli PabB determined in two different crystal forms, each at 2.0 A resolution. The 453-residue monomeric PabB has a complex alpha/beta fold which is similar to that seen in the structures of homologous, oligomeric TrpE subunits of several anthranilate synthases of microbial origin. A comparison of the structures of these two classes of chorismate-utilizing enzymes provides a rationale for the differences in quaternary structures seen for these enzymes, and indicates that the weak or transient association of PabB with PabA during catalysis stems at least partly from a limited interface for protein interactions. Additional analyses of the structures enabled the tentative identification of the active site of PabB, which contains a number of residues implicated from previous biochemical and genetic studies to be essential for activity. Differences in the structures determined from phosphate- and formate-grown crystals, and the location of an adventitious formate ion, suggest that conformational changes in loop regions adjacent to the active site may be needed for catalysis. A surprising finding in the structure of PabB was the presence of a tryptophan molecule deeply embedded in a binding pocket that is analogous to the regulatory site in the TrpE subunits of the anthranilate synthases. The strongly bound ligand, which cannot be dissociated without denaturation of PabB, may play a structural role in the enzyme since there is no effect of tryptophan on the enzymic synthesis of aminodeoxychorismate. Extensive sequence similarity in the tryptophan-binding pocket among several other chorismate-utilizing enzymes, including isochorismate synthase, suggests that they too may bind tryptophan for structural integrity, and corroborates early ideas on the evolution of this interesting enzyme family.

In Experiment 1, the effect of repeated injections of 2.5, 5.0, or 10.0 mg/kg of chlordiazepoxide (CDP) on food intake and body weight was studied in rats on an activity anorexia (AA) regimen. For several days before CDP testing began, rats lived in activity wheels and had one 60-min meal per day. During CDP testing, this regimen continued except that each rat was injected with an appropriate dose of CDP or saline 30 min before each meal. CDP enhanced food intake; 5.0 mg/kg seemed most effective. However, the CDP-induced increase in eating did not noticeably stem weight loss. In Experiment 2, after several days of AA training, CDP (5.0 mg/kg) was tested under less severe conditions; food remained restricted, but access to the wheels was discontinued. Rats given CDP ate more and gained more weight than controls. These findings suggest that benzodiazepines such as CDP may help in treating anorexia nervosa and other anorectic conditions in humans.

The structure of the tetradeca-(3-fluorotyrosyl) M1-1 GSH transferase (3-FTyr GSH transferase), a protein in which tyrosine residues are globally substituted by 3-fluorotyrosines has been determined at 2.2 A resolution. This variant was produced to study the effect on the enzymatic mechanism and the structure was undertaken to assess how the presence of the 3-fluorotyrosyl residue influences the protein conformation and hence its function. Although fluorinated amino acid residues have frequently been used in biochemical and NMR investigations of proteins, no structure of a protein that has been globally substituted with a fluorinated amino acid has previously been reported. Thus, this structure represents the first crystal structure of such a protein containing a library of 14 (28 crystallographically distinct) microenvironments from which the nature of the interactions of fluorine atoms with the rest of the protein can be evaluated. Numerous conformational changes are observed in the protein structure as a result of substitution of 3-fluorotyrosine for tyrosine. The results of the comparison of the crystal structure of the fluorinated protein with the native enzyme reveal that conformational changes are observed for most of the 3-fluorotyrosines. The largest differences are seen for residues where the fluorine, the OH, or both are directly involved in interactions with other regions of the protein or with a symmetry-related molecule. The fluorine atoms of the 3-fluorotyrosine interact primarily through hydrogen bonds with other residues and water molecules. In several cases, the conformation of a 3-fluorotyrosine is different in one of the monomers of the enzyme from that observed in the other, including different hydrogen-bonding patterns. Altered conformations can be related to differences in the crystal packing interactions of the two monomers in the asymmetric unit. The fluorine atom on the active-site Tyr6 is located near the S atom of the thioether product (9R,10R)-9-(S-glutathionyl)-10-hydroxy-9,10-dihydrophenanthrene and creates a different pattern of interactions between 3-fluorotyrosine 6 and the S atom. Studies of these interactions help explain why 3-FTyr GSH transferase exhibits spectral and kinetic properties distinct from the native GSH transferase., (Copyright 1998 Academic Press)

The catalytic characteristics and structure of the M1-1 isoenzyme of rat glutathione (GSH) transferase in which all four tryptophan residues in each monomer are replaced with 5-fluorotryptophan are described. The fluorine-for-hydrogen substitution does not change the interaction of the enzyme with GSH even though two tryptophan residues (Trp7 and Trp45) are involved in direct hydrogen-bonding interactions with the substrate. The rate constants for association and dissociation of the peptide, measured by stopped-flow spectrometry, remain unchanged by the unnatural amino acid. The 5-FTrp-substituted enzyme exhibits a kcat of 73 s-1 as compared to 18 s-1 for the native enzyme toward 1-chloro-2,4-dinitrobenzene. That the increase in the turnover number is due to an enhanced rate of product release in the mutant is confirmed by the kinetics of the approach to equilibrium for binding of the product. The crystal structure of the 5-FTrp-containing enzyme was solved at a resolution of 2.0 A by difference Fourier techniques. The structure reveals local conformational changes in the structural elements that define the approach to the active site which are attributed to steric interactions of the fluorine atoms associated with 5-FTrp146 and 5-FTrp214 in domain II. These changes appear to result in the enhanced rate of product release. This structure represents the first of a protein substituted with 5-fluorotryptophan.

Complex structures of a naturally occurring variant of human class pi glutathione S-transferase 1-1 (hGSTP1-1) with either S-hexylglutathione or (9R,10R)-9-(S-glutathionyl)-10-hydroxy-9, 10-dihydrophenanthrene [(9R,10R)-GSPhen] have been determined at resolutions of 1.8 and 1.9 A, respectively. The crystal structures reveal that the xenobiotic substrate-binding site (H-site) is located at a position similar to that observed in class mu GST 1-1 from rat liver (rGSTM1-1). In rGSTM1-1, the H-site is a hydrophobic cavity defined by the side chains of Y6, W7, V9, L12, I111, Y115, F208, and S209. In hGSTP1-1, the cavity is approximately half hydrophobic and half hydrophilic and is defined by the side chains of Y7, F8, V10, R13, V104, Y108, N204, and G205 and five water molecules. A hydrogen bond network connects the five water molecules and the side chains of R13 and N204. V104 is positioned such that the introduction of a methyl group (the result of the V104I mutation) disturbs the H-site water structure and alters the substrate-binding properties of the isozyme. The hydroxyl group of Y7 forms a hydrogen bond (3.2 A) with the sulfur atom of the product. There is a short hydrogen bond (2.5 A) between Y108 (OH) and (9R, 10R)-GSPhen (O5), indicating the hydroxyl group of Y108 as an electrophilic participant in the addition of glutathione to epoxides. An N-(2-hydroxethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) molecule is found in the cavity between beta2 and alphaI. The location and properties of this HEPES-binding site fit a possible non-substrate-binding site that is involved in noncompetitive inhibition of the enzyme.

The activation of the thiol of glutathione (GSH) bound in the active site of the class mu glutathione transferase M1-1 from rat involves a hydrogen-bonding network that includes a direct (first-sphere) interaction between the hydroxyl group of Y6 and the sulfur of GSH and second-sphere interactions involving a hydrogen bond between the main-chain amide N-H of L12 and the hydroxyl group of Y6 and an on-face hydrogen bond between the hydroxyl group of T13 and the pi-electron cloud of Y6 (i.e., T13-OH---pi-Y6-OH--- -SG). The functions of these hydrogen bonds have been examined with a combination of site-specific mutagenesis and X-ray crystallography. The hydroxyl group of Y6 has a normal pKa of about 10 even though it is shielded from solvent and is in a largely hydrophobic environment. The apparent pKa of GSH in the binary Y6F.GSH complex is increased by 1.6 log units, and the reactivity of the enzyme-bound nucleophile is reduced. The catalytic properties of the Y6L mutant are identical to those of Y6F, suggesting that the weakly polar on-edge interaction between the aromatic ring and sulfur has no influence on catalysis. The refined three-dimensional structure of the Y6F mutant in complex with GSH shows no major structural perturbation of the protein other than a change in the coordination environment of the sulfur. Removal of the second-sphere influence of the on-face hydrogen bond between the hydroxyl groups T13 as in the T13V and T13A mutants elevates the pKa of enzyme-bound GSH by about 0.7 pKa units. Crystal structures of these mutants show that structural changes in the active site are minor and suggest that the changes in pKa of E.GSH are due to the presence or absence of the on-face hydrogen bond. The T13S mutant has a completely different side-chain hydrogen-bonding geometry than T13 in the native enzyme and catalytic properties similar to the T13A and T13V mutants consistent with the absence of an on-face hydrogen bond. The gamma-methyl group of T13 is essential in enforcing the on-face hydrogen bond geometry and preventing the hydroxyl group from forming more favorable conventional hydrogen bonds.

A blind trial of 31 compounds was conducted in order to evaluate the intravitelline injection technique as a predictor of developmental toxicity. Rat conceptuses were explanted on day 9 of gestation and cultured until 11.5 days, at which time the compounds were injected into the vitelline circulation of the yolk sac. The embryos were then cultured for a further 24 hr after which time they were assessed on 14 parameters and assigned to one of three categories based on predetermined criteria. The results were then compared with the classifications previously allocated using data derived from in vitro studies. The compounds were examples of non-teratogens, non-cytotoxic teratogens and cytotoxic teratogens. Of the 31 compounds, 23 gave the predicted result, three non-teratogens being false positives and five teratogens having no effect in this system. The results therefore demonstrate the ability of the technique to distinguish between the three categories of compound, displaying a specificity of 75%, a sensitivity of 74% and an overall accuracy of 74.5%. Furthermore, it allowed the differentiation of active and inactive analogues of four paired compounds. This method may have applications as a teratogenicity screen or in the elucidation of the mechanism of action of teratogenic compounds.

A preliminary study of a novel developmental toxicity screen has been carried out. The technique involves the direct injection into the vitelline circulation of the 11.5-day rat conceptus, by-passing the metabolically active visceral yolk sac. The evaluation was performed blind using four coded model compounds: sulphanilamide (non-cytotoxic, non-teratogen), retinoic acid (teratogen) and methotrexate and cyclophosphamide (both cytotoxic teratogens). Seven parameters of teratogenicity and cytotoxicity were measured (yolk sac diameter, crown-rump length, somite number, yolk sac protein, yolk sac DNA, embryo protein, embryo DNA) and morphological abnormalities were also noted. The results showed that this technique successfully identified the developmental toxins and, moreover, differentiated between teratogens and cytotoxic teratogens. Additionally, the results show that methotrexate and cyclophosphamide produced an effect without prior exogenous activation as is necessary in other in vitro tests.

The results are presented on an interlaboratory study to validate the Micromass assay by testing compounds under code using a similar protocol, with an assessment of results for 25 compounds tested without S-9 mix. Four of these were co-tested with S-9 mix. Three separate sets of criteria have been proposed (Flint, 1986 and 1987; Flint and Orton, 1984) for interpreting the results for teratogenic hazard from in vitro data using IC(50) values: (i) the '< 500 mug/ml rule', (ii) the '< 50 mug/ml rule' and (iii) the 'specific inhibition of cell differentiation 2-fold rule'. The data were decoded and assessed using the criteria for their sensitivity, specificity and accuracy. Using the '< 500 mug/ml rule' a higher sensitivity was obtained but at the expense of a high false positive frequency (50%). Conversely, the '2-fold rule' gave a high specificity with only 10% false positives. Diphenhydramine and furazolidone gave false positive responses; the teratogens beta-aminopropionitrile and methotrexate were not detected. Selective inhibition of cell differentiation was related to high potential teratogenicity. In the application of the test, it is suggested that if there was an indication of teratogenic hazard using the '2-fold rule' the compound should be rejected without recourse to animal testing. In its present form the assay cannot be used to unequivocally identify non-teratogens.

A spin label analogue of compound 48/80 has been synthesized and its binding to purified rat peritoneal mast cells has been studied by electron spin resonance spectroscopy. The spin label analogue (SL-48/80) had almost identical biological activity to unlabeled compound 48/80. SL-48/80 was used to estimate the number of binding sites per cell on normal mast cells (7.25 X 10(10)), on mast cells deactivated by sodium azide and 2-deoxyglucose or by heating to 46 degrees for 30 min (1 X 10(10)) and cells from animals actively-sensitized to ovalbumin (5.2 X 10(10)). SL-48/80 was also shown to bind to isolated mast cell granules. Differences in the binding properties of mast cells after the different treatments are related to their surface topography as seen by scanning electron microscopy, and the contribution of the granules to the number of binding sites is discussed.

A system is described in which cells are perfused continuously with appropriate medium while simultaneously recording any change in cell morphology together with assay of released products (enzymes and mediators) following immunologic or non-immunologic perturbation. The released products in the perfusate are monitored continuously by automated fluorimetric assay procedures. The system is particularly applicable to investigate or toxicological studies of isolated mast cells or cultured macrophages.

Macrophages were exposed to lignocaine in perifused and non-perifused cultures using media with or without foetal calf serum. Effects on morphology, viability, phagocytosis and the release of enzymes were assessed. During the period of contact with lignocaine there was a selective release of beta-glucuronidase. After washing, enzyme release continued over a period of 7 hours and, in the absence of foetal calf serum, a decrease in the total beta-glucuronidase content was found in non-perifused cultures. Although lignocaine-treated cells phagocytosed particulates the rate of enzyme release was reduced compared with normal cells when subsequently exposed to quartz.

The effects of zinc pyrithione (ZnPTO) were studied in a series of in vitro tests to determine whether its mode of action is primarily cytostatic or cytotoxic. Sodium pyrithione (NaPTO) was also studied, to check that pyrithione was the active moiety, and the known cytostatic chemical hydroxyurea was included for comparison. ZnPTO had a reversible inhibitory effect on the growth of BHK 21 cells at 0.1 microgram/ml, but had a rapid, irreversible inhibitory effect at 1 microgram/ml associated with cell rounding and detachment. NaPTO produced a similar effect but hydroxyurea produced an essentially reversible inhibition even at a dose well above that producing complete inhibition. ZnPTO and NaPTO both caused contraction, rounding and blebbing of BHK 21 cells in perfusion-chamber tests, at higher levels (1 and 10 micrograms/ml) than required for growth inhibition, but only 10 micrograms ZnPTO/ml caused lactate dehydrogenase (LDH) release. Hydroxyurea had no effects in these tests. ZnPTO and NaPTO also reduced the survival of Chinese hamster V79 cells sharply over a narrow dose range (0.01-0.03 microgram/ml), but the effect of hydroxyurea was not as sharp and occurred at much higher doses. All three showed elements of cytostasis and cytotoxicity as demonstrated by analysis of the relationship between survival and colony area. Of the three, only ZnPTO (at greater than or equal to 5 micrograms/ml) caused significant LDH release from the cells, though both ZnPTO and NaPTO (at 0.1-1 microgram/ml) inhibited cell growth as indicated by total LDH values. In studies with rat peritoneal mast cells, ZnPTO and NaPTO (at 10 ng/ml) both suppressed histamine release induced by 48/80 or Ca ionophore A23187, though neither caused histamine release directly. The combined results of these tests show that ZnPTO is primarily cytotoxic, rather than cytostatic.

An automated assay suitable for the extraction and simultaneous fluorimetric determination of histamine and 5-hydroxytryptamine (5-HT) released from rat isolated peritoneal mast cells is described. The method gave good reproducibility and accuracy in use and required a sample volume of 0.5 ml for assay at a sampling rate of 30/h. The system was evaluated by assessment of its performance in the determination of histamine and 5-HT released from rat mast cells in response to compound 48/80. The detection limit for the procedure was 2 ng 5-HT base/ml and 20 ng histamine base/ml.