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Certain species of pathogenic bacteria damage tissues by secreting cholesterol-dependent cytolysins, which form pores in the plasma membranes of animal cells. However, reducing cholesterol protects cells against these cytolysins. As the first committed step of cholesterol biosynthesis is catalyzed by squalene synthase, we explored whether inhibiting this enzyme protected cells against cholesterol-dependent cytolysins. We first synthesized 22 different nitrogen-containing bisphosphonate molecules that were designed to inhibit squalene synthase. Squalene synthase inhibition was quantified using a cell-free enzyme assay, and validated by computer modeling of bisphosphonate molecules binding to squalene synthase. The bisphosphonates were then screened for their ability to protect HeLa cells against the damage caused by the cholesterol-dependent cytolysin, pyolysin. The most effective bisphosphonate reduced pyolysin-induced leakage of lactate dehydrogenase into cell supernatants by >80%, and reduced pyolysin-induced cytolysis from >75% to

Albaflavenone, a tricyclic sesquiterpene antibiotic, is biosynthesized in Streptomyces coelicolor A3(2) by enzymes encoded in a two-gene operon. Initially, sesquiterpene cyclase catalyzes the cyclization of farnesyl diphosphate to the terpenoid epi-isozizaene, which is oxidized to the final albaflavenone by cytochrome P450 (CYP)170A1. Additionally, this CYP is a bifunctional enzyme, being able to also generate farnesene isomers from farnesyl diphosphate, owing to a terpene synthase active site moonlighting on the CYP molecule. To explore the functionality of this operon in other streptomycetes, we have examined culture extracts by GC/MS and established the presence of albaflavenone in five Streptomyces species. Bioinformatics examination of the predicted CYP170 primary amino acid sequences revealed substitutions in the CYP terpene synthase active site. To examine whether the terpene synthase site was catalytically active in another CYP170, we characterized the least related CYP170 orthologue from Streptomyces albus (CYP170B1). Following expression and purification, CYP170B1 showed a normal reduced CO difference spectrum at 450 nm, in contrast to the unusual 440-nm peak observed for S. coelicolor A3(2) CYP170A1. CYP170B1 can catalyze the conversion of epi-isozizaene to albaflavenone, but was unable to catalyze the conversion of farnesyl diphosphate to farnesene. Molecular modeling with our crystal structure of CYP170A1 suggests that the absence of key amino acids for binding the essential terpene synthase cofactor Mg(2+) may be the explanation for the loss of CYP170B1 bifunctionality.

Aquaporins and aquaglyceroporins mediate the transport of water and solutes across biological membranes. Saccharomyces cerevisiae Fps1 is an aquaglyceroporin that mediates controlled glycerol export during osmoregulation. The transport function of Fps1 is rapidly regulated by osmotic changes in an apparently unique way and distinct regions within the long N- and C-terminal extensions are needed for this regulation. In order to learn more about the mechanisms that control Fps1 we have set up a genetic screen for hyperactive Fps1 and isolated mutations in 14 distinct residues, all facing the inside of the cell. Five of the residues lie within the previously characterized N-terminal regulatory domain and two mutations are located within the approach to the first transmembrane domain. Three mutations cause truncation of the C-terminus, confirming previous studies on the importance of this region for channel control. Furthermore, the novel mutations identify two conserved residues in the channel-forming B-loop as critical for channel control. Structural modelling-based rationalization of the observed mutations supports the notion that the N-terminal regulatory domain and the B-loop could interact in channel control. Our findings provide a framework for further genetic and structural analysis to better understand the mechanism that controls Fps1 function by osmotic changes.