John W. Tomsho

Microtubule-assisted mechanism for functional metabolic macromolecular complex formation

Evidence has been presented for a metabolic multienzyme complex, the purinosome, that participates in de novo purine biosynthesis to form clusters in the cytoplasm of living cells under purine-depleted conditions. Here we identified, using fluorescent live cell imaging, that a microtubule network appears to physically control the spatial distribution of purinosomes in the cytoplasm. Application of a cell-based assay measuring the rate of de novo purine biosynthesis confirmed that the metabolic activity of purinosomes was significantly suppressed in the absence of microtubules. Collectively, we propose a microtubule-assisted mechanism for functional purinosome formation in HeLa cells.

Elucidation of the Mechanism of the Reaction between Phenylboronic Acid and a Model Diol, Alizarin Red S

In this work, the reaction between phenylboronic acid and the diol-containing, fluorescent dye Alizarin Red S (ARS) was probed. Fluorescence titrations, (11)B NMR measurements, and both pre- and steady-state kinetic experiments were used for the characterization of this reaction over a large pH range (4-10.5). It was shown that ARS preferentially reacted with the boronic (neutral, trigonal) form of phenylboronic acid; however, the boronate (anionic, tetrahedral) form was also reactive. All in all, four reactant species were implicated in the formation of four different adduct species. The rate of a given adduct formation depended on the combination of the solution pH and the pK(a)s of both ARS and the arylboronic acid. The reaction was found to proceed in two distinct kinetic steps with the products and starting materials in facile exchange. In addition, the elucidation of the mechanism indicated the presence of two fluorescent products with the structure of the major contributor differing from what had been cited in the literature.

Discovery of antibacterial cyclic peptides that inhibit the ClpXP protease

A method to rapidly screen libraries of cyclic peptides in vivo for molecules with biological activity has been developed and used to isolate cyclic peptide inhibitors of the ClpXP protease. Fluorescence activated cell sorting was used in conjunction with a fluorescent reporter to isolate cyclic peptides that inhibit the proteolysis of tmRNA‐tagged proteins in Escherichia coli. Inhibitors shared little sequence similarity and interfered with unexpected steps in the ClpXP mechanism in vitro. One cyclic peptide, IXP1, inhibited the degradation of unrelated ClpXP substrates and has bactericidal activity when added to growing cultures of Caulobacter crescentus, a model organism that requires ClpXP activity for viability. The screen used here could be adapted to identify cyclic peptide inhibitors of any enzyme that can be expressed in E. coli in conjunction with a fluorescent reporter.

Examination of the reactivity of benzoxaboroles and related compounds with a cis-diol.

Benzoxaboroles have been emerging as an interesting and useful scaffold in drug discovery due to their apparently unique reactivity toward diols under physiological conditions. In this work, the reaction of benzoxaborole with the diol-containing, fluorescent dye Alizarin Red S is probed. Steady-state and presteady-state experiments have been conducted for the characterization of the reactions over a wide range of pH. Results indicate that Alizarin Red S reacts with both the boronic (neutral, trigonal) form as well as the boronate (anionic, tetrahedral) form of benzoxaborole in a reaction largely analogous to that previously determined for the simple phenylboronic acid. However, in certain key aspects, the reactivity of the benzoxaborole was found to differ from that of simple phenylboronic acid. The structural origin of these differences has been explored by examination of compounds related to both benzoxaborole and phenylboronic acid. These results may be applied to rational drug discovery efforts aimed at expanding the use of benzoxaboroles in medicine.

Concentration-Dependent Processivity of Multiple Glutamate Ligations Catalyzed by Folylpoly-γ-glutamate Synthetase

Folylpoly-gamma-glutamate synthetase (FPGS, EC 6.3.2.17) is an ATP-dependent ligase that catalyzes formation of poly-gamma-glutamate derivatives of reduced folates and antifolates such as methotrexate and 5,10-dideaza-5,6,7,8-tetrahydrofolate (DDAH 4PteGlu 1). While the chemical mechanism of the reaction catalyzed by FPGS is known, it is unknown whether single or multiple glutamate residues are added following each folate binding event. A very sensitive high-performance liquid chromatography method has been used to analyze the multiple ligation reactions onto radiolabeled DDAH 4PteGlu 1 catalyzed by FPGS to distinguish between distributive or processive mechanisms of catalysis. Reaction time courses, substrate trapping, and pulse-chase experiments were used to assess folate release during multiple glutamate additions. Together, the results of these experiments indicate that hFPGS can catalyze the processive addition of approximately four glutamate residues to DDAH 4PteGlu 1. The degree of processivity was determined to be dependent on the concentration of the folate substrate, thus suggesting a mechanism for the regulation of folate polyglutamate synthesis in cells.

Identification of Inhibitors of a Bacterial Sigma Factor Using a New High-Throughput Screening Assay

ABSTRACT Gram-negative bacteria are formidable pathogens because their cell envelope presents an adaptable barrier to environmental and host-mediated challenges. The stress response pathway controlled by the alternative sigma factor σE is critical for maintenance of the cell envelope. Because σE is required for the virulence or viability of several Gram-negative pathogens, it might be a useful target for antibiotic development. To determine if small molecules can inhibit the σE pathway, and to permit high-throughput screening for antibiotic lead compounds, a σE activity assay that is compatible with high-throughput screening was developed and validated. The screen employs a biological assay with positive readout. An Escherichia coli strain was engineered to express yellow fluorescent protein (YFP) under negative regulation by the σE pathway, such that inhibitors of the pathway increase the production of YFP. To validate the screen, the reporter strain was used to identify σE pathway inhibitors from a library of cyclic peptides. Biochemical characterization of one of the inhibitory cyclic peptides showed that it binds σE, inhibits RNA polymerase holoenzyme formation, and inhibits σE-dependent transcription in vitro. These results demonstrate that alternative sigma factors can be inhibited by small molecules and enable high-throughput screening for inhibitors of the σE pathway.

Inhibition of human folylpolyglutamate synthetase by diastereomeric phosphinic acid mimics of the tetrahedral intermediate.

Phosphorus-containing pseudopeptides, racemic at the C-terminal alpha-carbon, are potent mechanism-based inhibitors of folylpolyglutamate synthetase (FPGS). They are mimics of the tetrahedral intermediate postulated to form during FPGS-catalyzed biosynthesis of poly(gamma-l-glutamates). In the present paper, the FPGS inhibitory activity of each diastereomer coupled to three heterocycles is reported. The high R(f) pseudopeptide containing the 5,10-dideazatetrahydropteroyl (DDAH(4)Pte) heterocycle is most potent (K(is) = 1.7 nM). While the heterocyclic portion affects absolute FPGS inhibitory potency, the high R(f) species is more potent in each pair containing the same heterocycle. This species presumably has the same stereochemistry as the natural folate polyglutamate, i.e., (l-Glu-gamma-l-Glu). Unexpectedly, the low R(f) (presumed l-Glu-gamma-d-Glu) species are only slightly less potent (<30-fold) than their diastereomers. Further study of this phenomenon comparing l-Glu-gamma-l-Glu and l-Glu-gamma-d-Glu dipeptide-containing FPGS substrates shows that <1% contamination of commercial d-Glu precursors by l-Glu may give misleading information if l-Glu-gamma-l-Glu substrates have low K(m) values.

Unnatural translation initiation.

Protein translation in nature always begins with an initiator transfer RNA (tRNA) carrying the amino acid methionine. This was circumvented in vitro with a reconstituted translation system utilizing initiator tRNA synthetically mischarged with the other natural amino acids. In addition, it was determined that this system could accommodate these non-methionine amino acids containing various N-alpha-acyl groups, many of which are useful for post-translational modification such as peptide cyclization.