Natural products provide an unparalleled source of chemical scaffolds with diverse biological activities and have profoundly impacted antimicrobial drug discovery. To further explore the full potential of their chemical diversity, we survey natural products for antifungal, target-specific inhibitors by using a chemical-genetic approach adapted to the human fungal pathogen Candida albicans and demonstrate that natural-product fermentation extracts can be mechanistically annotated according to heterozygote strain responses. Applying this approach, we report the discovery and characterization of a natural product, parnafungin, which we demonstrate, by both biochemical and genetic means, to inhibit poly(A) polymerase. Parnafungin displays potent and broad spectrum activity against diverse, clinically relevant fungal pathogens and reduces fungal burden in a murine model of disseminated candidiasis. Thus, mechanism-of-action determination of crude fermentation extracts by chemical-genetic profiling brings a powerful strategy to natural-product-based drug discovery.

Mechanism-of-action (MOA) studies of bioactive compounds are fundamental to drug discovery. However, in vitro studies alone may not recapitulate a compound's MOA in whole cells. Here, we apply a chemogenomics approach in Candida albicans to evaluate compounds affecting purine metabolism. They include the IMP dehydrogenase inhibitors mycophenolic acid and mizoribine and the previously reported GMP synthase inhibitors acivicin and 6-diazo-5-oxo-L-norleucine (DON). We report important aspects of their whole-cell activity, including their primary target, off-target activity, and drug metabolism. Further, we describe ECC1385, an inhibitor of GMP synthase, and provide biochemical and genetic evidence supporting its MOA to be distinct from acivicin or DON. Importantly, GMP synthase activity is conditionally essential in C. albicans and Aspergillus fumigatus and is required for virulence of both pathogens, thus constituting an unexpected antifungal target.

2-Cys peroxiredoxin (2-Cys Prx) is a large group of proteins that participate in cell proliferation, differentiation, apoptosis, and photosynthesis. In the prevailing view, this ubiquitous peroxidase poises the concentration of H2O2 and, in so doing, regulates signal transduction pathways or protects macromolecules against oxidative damage. Here, we describe the first purification of 2-Cys Prx from higher plants and subsequently we show that the native and the recombinant forms of rapeseed leaves stimulate the activity of chloroplast fructose-1,6-bisphosphatase (CFBPase), a key enzyme of the photosynthetic CO2 assimilation. The absence of reductants, the strict requirement of both fructose 1,6-bisphosphate and Ca2+, and the response of single mutants C174S and C179S CFBPase bring forward clear differences with the well-known stimulation mediated by reduced thioredoxin via the regulatory 170's loop of CFBPase. Taken together, these findings provide an unprecedented insight into chloroplast enzyme regulation wherein both 2-Cys Prx and the 170's loop of CFBPase exhibit novel functions.

(1 --> 6)-beta-D-glucan is a key cell wall component of Saccharomyces cerevisiae and Candida albicans. Many genes are known to affect the levels or structure of this glucan, but their roles and a molecular description of the synthesis of (1 --> 6)-beta-D-glucan remain to be established and a method to measure (1 --> 6)-beta-D-glucan synthase activity in vitro would provide an enabling tool. Here, conditions for the detection of in vitro synthesis of this polymer are described. Crude membrane preparations from S. cerevisiae were isolated, and incubated in the presence of UDP-glucose and GTP. With anti-(1 --> 6)-beta-D-glucan-specific antibodies, a time-dependent increase in the amount of this glucan was demonstrated in a dot-blot assay, or through an inhibition enzyme immunoassay. Antibody specificity was validated by competition experiments using pustulan, a (1 --> 6)-beta-D-glucan, laminarin, a (1 --> 3)-beta-D-glucan, yeast mannan and glycogen. The identity of the reaction product was also demonstrated by its sensitivity to a recombinant (1 --> 6)-beta-D-glucanase. Extracts from mutants in 10 genes with a wide range of altered cell wall (1 --> 6)-beta-D-glucan levels were assayed for in vitro synthesis of the polymer. A strong correlation of in vitro synthase activity with in vivo glucan levels was found, providing genetic support for the specificity of the assay. The basis for the GTP-dependence of the synthase reaction was studied. Extracts from rho2, rho3, rho4 and rho5 null mutants had wild-type in vitro activity. In contrast, Rho1p overproduction led to increased in vitro synthesis, implicating Rho1p in the regulation of (1 --> 6)-beta-D-glucan synthesis.

The COP9 signalosome (CSN) is a conserved protein complex with homologies to the lid subcomplex of the 26S proteasome [1]. It promotes cleavage of the Nedd8 conjugate (deneddylation) from the cullin component of SCIF ubiquitin ligases [2]. We provide evidence that cullin neddylation and deneddylation is highly dynamic, that its equilibrium can be effectively modulated by CSN, and that neddylation allows Cull to form larger protein complexes. CSN2 integrates into the CSN complex via its C-terminal region and its N-terminal half region is necessary for direct interaction with Cull. The polyclonal antibodies against CSN2 but not other CSN subunits cause accumulation of neddylated Cu11/Cu12 in HeLa cell extract, indicating that CSN2 is essential in cullin deneddylation. Further, CSN inhibits ubiquitination and degradation of the cyclin-dependent kinase inhibitor p27(klp1) in vitro. Microinjection of the CSN complex impeded the G1 cells from entering the S phase. Moreover, anti-CSN2 antibodies negate the CSN-dependent p27 stabilization and the G1/S blockage, suggesting that these functions require the deneddylation activity. We conclude that CSN inhibits SCF ubiquitin ligase activity in targeting p27 proteolysis and negatively regulates cell cycle at the G1 phase by promoting deneddylation of Cul1.

Chloroplast Fru-1,6-bisphosphatase is a key enzyme of the Benson-Calvin cycle for photosynthetic COZ assimilation in higher plants. Although the capacity to hydrolyze Fru-1,6bisP is low in dark-adapted chloroplasts, it increases upon illumination. Numerous authors have identified the Fd-thioredoxin system (Fd, Fd-thioredoxin reductase, thioredoxin) as the mediator in enzyme activation (Wolosiuk et al., 1993). On the other hand, the concerted action of a reductant, a sugar bisphosphate, a bivalent cation, and either co-solvents, chaotropic anions, or high hydrostatic pressure stimulate in vitro the enzyme activity. Thus, the replacement of reduced thioredoxin with nonphysiological modulators disclosed the importance of appropriate conformations not only in the activation process but also for the assembly of the nucleaiencoded Fru-1,6-bisphosphatase in the chloroplast stroma (Lallicora and Wolosiuk, 1990). To analyze these processes, we isolated and sequenced a cDNA coding for the chloroplast Fru-1,6-bisphosphatase from rapeseed (Brassica napus) (Table I). Polyadenylated mRNA, isolated from 30-d-old green leaves, was used for the synthesis of cDNA, which was subsequently cloned between NotIlSalI sites of pSPORTl (BRL). The cDNA library was screened for Fru-1,6-bisphosphatase expression with a polyclonal antibody raised in rabbits against the spinach chloroplast enzyme. In positive dones, the presence of cDNA inserts coding for the enzyme was confirmed in a Southem blot assay using a DNA probe coding for the wheat counterpart (kindly provided by Dr. T. Dyer). The largest cDNA insert obtained was used for sequencing.

Chloroplast thioredoxin-f functions efficiently in the light-dependent activation of chloroplast fructose-1, 6-bisphosphatase by reducing a specific disulfide bond located at the negatively charged domain of the enzyme. Around the nucleophile cysteine of the active site (-W-C-G-P-C-), chloroplast thioredoxin-f shows lower density of negative charges than the inefficient modulator Escherichia coli thioredoxin. To examine the contribution of long range electrostatic interactions to the thiol/disulfide exchange between protein-disulfide oxidoreductases and target proteins, we constructed three variants of E. coli thioredoxin in which an acidic (Glu-30) and a neutral residue (Leu-94) were replaced by lysines. After purification to homogeneity, the reduction of the unique disulfide bond by NADPH via NADP-thioredoxin reductase proceeded at similar rates for all variants. However, the conversion of cysteine residues back to cystine depended on the target protein. Insulin and difluoresceinthiocarbamyl-insulin oxidized the sulfhydryl groups of E30K and E30K/L94K mutants more effectively than those of wild type and L94K counterparts. Moreover, the affinity of E30K, L94K, and E30K/L94K E. coli thioredoxin for chloroplast fructose-1,6-bisphosphatase (A0.5 = 9, 7, and 3 microM, respectively) increased with the number of positive charges, and was higher than wild type thioredoxin (A0.5 = 33 microM), though still lower than that of thioredoxin-f (A0.5 = 0.9 microM). We also demonstrated that shielding of electrostatic interactions with high salt concentrations not only brings the A0.5 for all bacterial variants to a limiting value of approximately 9 microM but also increases the A0.5 of chloroplast thioredoxin-f. While negatively charged chloroplast fructose-1,6-bisphosphatase (pI = 4.9) readily interacted with mutant thioredoxins, the reduction rate of rapeseed napin (pI = 11.2) diminished with the number of novel lysine residues. These findings suggest that the electrostatic interactions between thioredoxin and (some of) its target proteins controls the formation of the binary noncovalent complex needed for the subsequent thiol/disulfide exchange.

In higher plants, light enhances the activity of chloroplast fructose-1,6-bisphosphatase via a cascade of thiol/disulfide exchanges. We have examined the structural and functional role of seven conserved cysteine residues in the rapeseed (Brassica napus) enzyme by site-directed mutagenesis. After lysis ofEscherichia colicells, C53S and C191S variants partitioned mainly in the insoluble fraction whereas C96S, C157S, C174S, C179S, and C307S mutants were soluble. Homogeneous preparations of the latter hydrolyzed fructose 1,6-bisphosphate at similar rates in the presence of 10 mM Mg2+but only C157S, C174S and C179S mutants were both efficient catalysts at 1 mM Mg2+and nearly insensitive to dithiothreitol. These results demonstrate the contribution of Cys53 and Cys191 to the stability of the enzyme and the participation of Cys157, Cys174 and Cys179 in the reductive process responsive of the light-dependent regulation. Given that mutations at Cys96 and Cys307 neither destabilize the enzyme nor affect the reductive modulation, their function remains unknown.

In chloroplasts, the light-modulated fructose-1,6-bisphosphatase catalyzes the formation of fructose 6-bisphosphate for the photosynthetic assimilation of CO2 and the biosynthesis of starch. We report here the construction of a plasmid for the production of chloroplast fructose-1,6-bisphosphatase in a bacterial system and the subsequent purification to homogeneity of the genetically engineered enzyme. To this end, a DNA sequence that coded for chloroplast fructose-1,6-bisphosphatase of rapeseed (Brassica napus) leaves was successively amplified by PCR, ligated into the Ndel/EcoRI restriction site of the expression vector pET22b, and introduced into Escherichia coli cells. When gene expression was induced by isopropyl-β-D-thiogalactopyranoside, supernatants of cell lysates were extremely active in the hydrolysis of fructose 1,6-bisphosphate. Partitioning bacterial soluble proteins by ammonium sulfate followed by anion exchange chromatography yielded 10 mg of homogeneous enzyme per 1 of culture. Congruent with a preparation devoid of contaminating proteins, the Edman degradation evinced an unique N-terminal amino acid sequence [A-V-A-A-D-A-T-A-E-T-K-P-]. Gel filtration experiments and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the (recombinant) rapeseed chloroplast fructose-1,6-bisphosphatases was a tetramer [160 kDa] comprised of four identical subunits. Like other chloroplast fructose-1,6-bisphosphatases, the recombinant enzyme was inactive at 1 mM fructose 1,6-bisphosphate and 1 mM Mg(2+) but became fully active after an incubation in the presence of either 10 mM dithiothreitol or 1 mM dithiothreitol and chloroplast thioredoxin. However, at variance with counterparts isolated from higher plant leaves, the low activity observed in absence of reductants was not greatly enhanced by high concentrations of fructose 1,6-bisphosphate (3 mM) and Mg(2+) (10 mM). In the catalytic process, all chloroplast fructose-1,6-bisphosphatases had identical features; viz., the requirement of Mg(2+) as cofactor and the inhibition by Ca(2+). Thus, the procedure described here should prove useful for the structural and kinetic analysis of rapeseed chloroplast fructose-1,6-bisphosphatase in view that this enzyme was not isolated from leaves.

Chloroplast Fru-1,6-bisphosphatase is a key enzyme of the Benson-Calvin cycle for photosynthetic COZ assimilation in higher plants. Although the capacity to hydrolyze Fru-1,6bisP is low in dark-adapted chloroplasts, it increases upon illumination. Numerous authors have identified the Fd-thioredoxin system (Fd, Fd-thioredoxin reductase, thioredoxin) as the mediator in enzyme activation (Wolosiuk et al., 1993). On the other hand, the concerted action of a reductant, a sugar bisphosphate, a bivalent cation, and either co-solvents, chaotropic anions, or high hydrostatic pressure stimulate in vitro the enzyme activity. Thus, the replacement of reduced thioredoxin with nonphysiological modulators disclosed the importance of appropriate conformations not only in the activation process but also for the assembly of the nucleaiencoded Fru-1,6-bisphosphatase in the chloroplast stroma (Lallicora and Wolosiuk, 1990). To analyze these processes, we isolated and sequenced a cDNA coding for the chloroplast Fru-1,6-bisphosphatase from rapeseed (Brassica napus) (Table I). Polyadenylated mRNA, isolated from 30-d-old green leaves, was used for the synthesis of cDNA, which was subsequently cloned between NotIlSalI sites of pSPORTl (BRL). The cDNA library was screened for Fru-1,6-bisphosphatase expression with a polyclonal antibody raised in rabbits against the spinach chloroplast enzyme. In positive dones, the presence of cDNA inserts coding for the enzyme was confirmed in a Southem blot assay using a DNA probe coding for the wheat counterpart (kindly provided by Dr. T. Dyer). The largest cDNA insert obtained was used for sequencing.