The Pkc1-mediated cell wall integrity-signaling pathway is highly conserved in fungi and is essential for fungal growth. We thus explored the potential of targeting the Pkc1 protein kinase for developing broadspectrum fungicidal antifungal drugs through a Candida albicans Pkc1-based high-throughput screening. We discovered that cercosporamide, a broad-spectrum natural antifungal compound, but previously with an unknown mode of action, is actually a selective and highly potent fungal Pkc1 kinase inhibitor. This finding provides a molecular explanation for previous observations in which Saccharomyces cerevisiae cell wall mutants were found to be highly sensitive to cercosporamide. Indeed, S. cerevisiae mutant cells with reduced Pkc1 kinase activity become hypersensitive to cercosporamide, and this sensitivity can be suppressed under high-osmotic growth conditions. Together, the results demonstrate that cercosporamide acts selectively on Pkc1 kinase and, thus, they provide a molecular mechanism for its antifungal activity. Furthermore, cercosporamide and a ␤-1,3-glucan synthase inhibitor echinocandin analog, by targeting two different key components of the cell wall biosynthesis pathway, are highly synergistic in their antifungal activities. The synergistic antifungal activity between Pkc1 kinase and ␤-1,3-glucan synthase inhibitors points to a potential highly effective combination therapy to treat fungal infections.

To understand the biochemical basis of resistance of bacteria to ␤-lactam antibiotics, we purified a penicillin-resistant penicillin-binding protein 2x (R-PBP2x) and a penicillin-sensitive PBP2x (S-PBP2x) enzyme of Streptococcus pneumoniae and characterized their transpeptidase activities, using a thioester analog of stem peptides as a substrate. A comparison of the k cat /K m values for the two purified enzymes (3,400 M ؊1 s ؊1 for S-PBP2x and 11.2 M ؊1 s ؊1 for R-PBP2x) suggests that they are significantly different kinetically. Implications of this finding are discussed. We also found that the two purified enzymes did not possess a detectable level of ␤-lactam hydrolytic activity. Finally, we show that the expression levels of both PBP2x enzymes were similar during different growth phases.

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Deacetoxycephalosporin C synthetase (expandase), which catalyzes ring expansion of penicillin N to deacetoxycephalosporin C (DAOC), has been stabilized in vitro and purified to near homogeneity from the industrially important fungus Cephalosporium acremonium. Throughout the purification, the expandase activity remained physically associated with and in a constant ratio of 7:1 to DAOC hydroxylase activity. The latter activity mediates hydroxylation of DAOC to deacetylcephalosporin C (DAC). The copurified expandase/hydroxylase appeared to be monomeric, with a molecular weight of 41,000 +/- 2,000 and an isoelectric point of 6.3 +/- 0.3. Both catalytic activities required alpha-ketoglutarate, Fe2+, and O2 and were stimulated by ascorbate, dithiothreitol, and ATP. The Fe2+ requirement was specific, and sulfhydryl groups in the purified protein were apparently essential for both ring expansion and hydroxylation. The kinetics and stoichiometry of DAOC/DAC formation from the expandase/hydroxylase-catalyzed reactions suggested that ring expansion of penicillin N preceded hydroxylation of DAOC.

AMP-activated protein kinase (AMPK) serves as an energy-sensing protein kinase that is activated by a variety of metabolic stresses that lower cellular energy levels. When activated, AMPK modulates a network of metabolic pathways that result in net increased substrate oxidation, generation of reduced nucleotide cofactors, and production of ATP. AMPK is activated by a high AMP:ATP ratio and phosphorylation on threonine 172 by an upstream kinase. Recent studies suggest that mechanisms that do not involve changes in adenine nucleotide levels can activate AMPK. Another sensor of the metabolic state of the cell is the NAD/NADH redox potential. To test whether the redox state might have an effect on AMPK activity, we examined the effect of ␤-NAD and NADH on this enzyme. The recombinant T172D-AMPK, which was mutated to mimic the phosphorylated state, was activated by ␤-NAD in a dose-dependent manner, whereas NADH inhibited its activity. We explored the effect of NADH on AMPK by systematically varying the concentrations of ATP, NADH, peptide substrate, and AMP. Based on our findings and established activation of AMPK by AMP, we proposed a model for the regulation by NADH. Key features of this model are as follows. (a) NADH has an apparent competitive behavior with respect to ATP and uncompetitive behavior with respect to AMP resulting in improved binding constant in the presence of AMP, and (b) the binding of the peptide is not significantly altered by NADH. In the absence of AMP, the binding constant of NADH becomes higher than physiologically relevant. We conclude that AMPK senses both components of cellular energy status, redox potential, and phosphorylation potential.The mammalian 5Ј-AMP-activated protein kinase (AMPK) 1 serves as an energy-sensing protein kinase. It is activated by a variety of metabolic stresses that lower cellular energy levels. These include exercise, nutrient starvation, ischemia/hypoxia, heat shock, and metabolic poisoning (1-4). AMPK is also activated by a number of pharmacological interventions. AICAR (5-aminoimidazole-4-carboxamide 1-b-D-ribonucleoside), which is converted to an AMP analog upon entering the cells, is a commonly used tool for activating AMPK (5). Leptin (6), adiponectin (7), metformin (8, 9), and rosiglitazone (8) activate AMPK through poorly characterized mechanisms that may not involve energy depletion. The activation of AMPK stimulates fatty acid oxidation, hexokinase activity, and uptake of glucose into skeletal muscle, which generates reduced nucleoside cofactors and ATP, and represses hepatic gluconeogenic and fatty acid synthesis enzymes, which consume them (10).AMPK is a heterotrimeric complex consisting of a catalytic (␣) subunit and two regulatory subunits (␤ and ␥) (11). The isoforms of all three subunits have been identified including two isoforms of the catalytic subunit, ␣ 1 and ␣ 2 (9), two of the regulatory subunits, ␤ 1 and ␤ 2 , and three of the regulatory subunits, ␥ 1 , ␥ 2 , and ␥ 3 (12). The formation of the trimeric complex is necessary for optimal kin...