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The opportunistic pathogen Pseudomonas aeruginosa has two complete acyl-homoserine lactone (acyl-HSL) signaling systems, LasR-LasI and RhlR-RhlI. LasI catalyzes the synthesis of N-3-oxododecanoyl homoserine lactone (3OC12-HSL), and LasR is a transcription factor that requires 3OC12-HSL as a ligand. RhlI catalyzes the synthesis of N-butanoyl homoserine lactone (C 4 ), and RhlR is a transcription factor that responds to C 4 . LasR and RhlR control the transcription of hundreds of P. aeruginosa genes, many of which are critical virulence determinants, and LasR is required for RhlR function. We developed an ultra-high-throughput cell-based assay to screen a library of approximately 200,000 compounds for inhibitors of LasR-dependent gene expression. Although the library contained a large variety of chemical structures, the two best inhibitors resembled the acyl-homoserine lactone molecule that normally binds to LasR. One compound, a tetrazole with a 12-carbon alkyl tail designated PD12, had a 50% inhibitory concentration (IC 50 ) of 30 nM. The second compound, V-06-018, had an IC 50 of 10 M and is a phenyl ring with a 12-carbon alkyl tail. A microarray analysis showed that both compounds were general inhibitors of quorum sensing, i.e., the expression levels of most LasR-dependent genes were affected. Both compounds also inhibited the production of two quorumsensing-dependent virulence factors, elastase and pyocyanin. These compounds should be useful for studies of LasR-dependent gene regulation and might serve as scaffolds for the identification of new quorum-sensing modulators.

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The pathogenic bacterium Pseudomonas aeruginosa uses acylhomoserine lactone quorum-sensing signals to coordinate the expression of a battery of virulence genes in a cascade of regulatory events. The quorum-sensing signal that triggers the cascade is N-3-oxo-dodecanoyl homoserine lactone (3OC12-HSL), which interacts with two signal receptor-transcription factors, LasR and QscR. This signal is base labile, and it is degraded by mammalian PON lactonases. We have identified a structurally unrelated triphenyl mimic of 3OC12-HSL that is base-insensitive and PON-resistant. The triphenyl mimic seems to interact specifically with LasR but not with QscR. In silico analysis suggests that the mimic fits into the 3OC12-HSL-binding site of LasR and makes key contacts with LasR. The triphenyl mimic is an excellent scaffold for developing quorum-sensing inhibitors, and its stability and potency make it ideal for biotechnology uses such as heterologous gene expression.

Polyhydroxyalkanoate synthase (PHA) from Chromatium Vinosum catalyzes the conversion of 3-hydroxybutyryl-CoA (HB-CoA) to polyhydroxybutyrate (PHB) and CoA. The synthase is composed of a ∼1:1 mixture of two subunits, PhaC and PhaE. Size-exclusion chromatography indicates that in solution PhaC and PhaE exist as large molecular weight aggregates. The holo-enzyme, PhaEC, has a specific activity of 150 units/mg. Each subunit was cloned, expressed, and purified as a (His) 6 -tagged construct. The PhaC-(His) 6 protein catalyzed polymerization with a specific activity of 0.9 unit/mg; the PhaE-(His) 6 protein was inactive (specific activity <0.001 unit/mg). Addition of PhaE-(His) 6 to PhaC-(His) 6 increased the activity several 100-fold. To investigate the priming step of the polymerization process, the PhaEC was incubated with a trimer of HB-CoA in which the terminal hydroxyl was replaced with tritium ( Sequencing by ion trap mass spectrometry showed that they were identical and that they each contained an altered cysteine (C149). One peptide contained the [ 3 H]-sT while the other two contained, in addition to the [ 3 H]-sT, one and two additional monomeric HBs, respectively. Mutation of C149 to alanine gave inactive synthase. The remaining two cysteines of PhaC, 292 and 130, were also mutated to alanine. The former had wild-type (wt) activity, while the latter had 0.004 wt % activity and was capable of making polymer. A mechanism is proposed in which PhaC contains all the elements essential for catalysis and the polymerization proceeds by covalent catalysis using C149 and potentially C130.Polyhydroxyalkanoates (PHAs 1 ) are polyoxoesters with properties that range from elastomers to thermoplastics (1-4). They are produced by a wide range of bacteria when they are placed in an environment of nutrient limitation (5). Copolymers of polyhydroxybutyrate (PHB) and polyhydroxyvalerate in the correct ratio have properties similar to the petrochemically based polypropylenes, the major component of bulk plastics (6). In 1997, the US market for thermoplastics was on the order of 40 million tons per year (7). PHAs have recently received much attention because they are biodegradable and can be generated from biorenewable sources: bacteria and plants (8). The major focus of many investigators is to make their production economically competitive with the polypropylenes. To achieve this goal, the requirements for the polymerization process need to be established. This paper focuses on the PHA synthase from Chromatium Vinosum which catalyzes the formation of PHB from 3-hydroxybutyryl-CoA (HB-CoA). Evidence is presented that two cysteines and covalent catalysis play an important role in the initiation and elongation of the polymerization process.PHA synthases from 20 organisms have now been identified. They have been divided into three classes based on their substrate specificity and subunit composition (9). The class I synthases, with the Ralstonia eutropha synthase as a prototype, are composed of a single polypeptide (∼65 k...

In patients chronically infected with hepatitis C virus (HCV) strains of genotype 1, rapid and dramatic antiviral activity has been observed with telaprevir (VX-950), a highly selective and potent inhibitor of the HCV NS3-4A serine protease. HCV variants with substitutions in the NS3 protease domain were observed in some patients during telaprevir dosing. In this study, purified protease domain proteins and reconstituted HCV subgenomic replicons were used for phenotypic characterization of many of these substitutions. V36A/M or T54A substitutions conferred less than eightfold resistance to telaprevir. Variants with double substitutions at Val 36 plus Thr 54 had ϳ20-fold resistance to telaprevir, and variants with double substitutions at Val 36 plus Arg 155 or Ala 156 had >40-fold resistance to telaprevir. An X-ray structure of the HCV strain H protease domain containing the V36M substitution in a cocomplex with an NS4A cofactor peptide was solved at a 2.4-Å resolution. Except for the side chain of Met 36 , the V36M variant structure is identical to that of the wild-type apoenzyme. The in vitro replication capacity of most variants was significantly lower than that of the wild-type replicon in cells, which is consistent with the impaired in vivo fitness estimated from telaprevir-dosed patients. Finally, the sensitivity of these replicon variants to alpha interferon or ribavirin remained unchanged compared to that of the wild-type.

No abstract

Telaprevir (VX-950) is a highly selective, potent inhibitor of the hepatitis C virus (HCV) NS3⅐4A serine protease. It has demonstrated strong antiviral activity in patients chronically infected with genotype 1 HCV when dosed alone or in combination with peginterferon alfa-2a. Substitutions of Arg 155 of the HCV NS3 protease domain have been previously detected in HCV isolates from some patients during telaprevir dosing. In this study, Arg 155 was replaced with various residues in genotype 1a protease domain proteins and in genotype 1b HCV subgenomic replicons. Characterization of both the purified enzymes and reconstituted replicon cells demonstrated that substitutions of Arg 155 with these residues conferred low level resistance to telaprevir (<25-fold). An x-ray structure of genotype 1a HCV protease domain with the R155K mutation, in a complex with an NS4A co-factor peptide, was determined at a resolution of 2.5 Å . The crystal structure of the R155K protease is essentially identical to that of the wild-type apoenzyme (Protein Data Bank code 1A1R) except for the side chain of mutated residue 155. Telaprevir was docked into the x-ray structure of the R155K protease, and modeling analysis suggests that the P2 group of telaprevir loses several hydrophobic contacts with the Lys 155 side chain. It was demonstrated that replicon cells containing substitutions at NS3 protease residue 155 remain fully sensitive to interferon ␣ or ribavirin. Finally, these variant replicons were shown to have reduced replication capacity compared with the wild-type HCV replicon in cells.