Robert P. Gibson

Tps1 regulates the pentose phosphate pathway, nitrogen metabolism and fungal virulence

Trehalose fulfils a wide variety of functions in cells, acting as a stress protectant, storage carbohydrate and compatible solute. Recent evidence, however, indicates that trehalose metabolism may exert important regulatory roles in the development of multicellular eukaryotes. Here, we show that in the plant pathogenic fungus Magnaporthe grisea trehalose‐6‐phosphate (T6P) synthase (Tps1) is responsible for regulating the pentose phosphate pathway, intracellular levels of NADPH and fungal virulence. Tps1 integrates glucose‐6‐phosphate (G6P) metabolism with nitrogen source utilisation, and thereby regulates the activity of nitrate reductase. Activity of Tps1 requires an associated regulator protein Tps3, which is also necessary for pathogenicity. Tps1 controls expression of the nitrogen metabolite repressor gene, NMR1, and is required for expression of virulence‐associated genes. Functional analysis of Tps1 indicates that its regulatory functions are associated with binding of G6P, but independent of Tps1 catalytic activity. Taken together, these results demonstrate that Tps1 is a central regulator for integration of carbon and nitrogen metabolism, and plays a pivotal role in the establishment of plant disease.

An NADPH-dependent genetic switch regulates plant infection by the rice blast fungus.

To cause rice blast disease, the fungus Magnaporthe oryzae breaches the tough outer cuticle of the rice leaf by using specialized infection structures called appressoria. These cells allow the fungus to invade the host plant and proliferate rapidly within leaf tissue. Here, we show that a unique NADPH-dependent genetic switch regulates plant infection in response to the changing nutritional and redox conditions encountered by the pathogen. The biosynthetic enzyme trehalose-6-phosphate synthase (Tps1) integrates control of glucose-6-phosphate metabolism and nitrogen source utilization by regulating the oxidative pentose phosphate pathway, the generation of NADPH, and the activity of nitrate reductase. We report that Tps1 directly binds to NADPH and, thereby, regulates a set of related transcriptional corepressors, comprising three proteins, Nmr1, Nmr2, and Nmr3, which can each bind NADP. Targeted deletion of any of the Nmr-encoding genes partially suppresses the nonpathogenic phenotype of a Δtps1 mutant. Tps1-dependent Nmr corepressors control the expression of a set of virulence-associated genes that are derepressed during appressorium-mediated plant infection. When considered together, these results suggest that initiation of rice blast disease by M. oryzae requires a regulatory mechanism involving an NADPH sensor protein, Tps1, a set of NADP-dependent transcriptional corepressors, and the nonconsuming interconversion of NADPH and NADP acting as signal transducer.

Mechanistic Insight into Enzymatic Glycosyl Transfer with Retention of Configuration through Analysis of Glycomimetic Inhibitors

Structural “valid”-ation: The mechanism of enzyme-catalyzed glycosyl transfer with retention of anomeric configuration continues to baffle, a situation compounded by the lack of insightful 3-D structures of ternary enzyme complexes. Synthesis and multi-dimensional kinetic analysis of validoxylamine derivatives are used to access the 3-D structure of a ternary complex (see picture; U=uridyl) providing insight into the geometry and donor–acceptor interplay at the glycosyl transfer site.

Characterization of Escherichia coli OtsA, a trehalose-6-phosphate synthase from glycosyltransferase family 20

The Ots gene cluster of Escherichia coli encodes the synthetic apparatus for the formation of alpha,alpha-1,1-trehalose, a non-reducing glucose disaccharide. The otsA gene encodes a trehalose-6-phosphate synthase, a glycosyltransferase which catalyses the synthesis of alpha,alpha-1,1-trehalose-6-phosphate from glucose-6-phosphate using a UDP-glucose donor. It has been classified into glycosyltransferase family GT-20 based upon amino-acid sequence similarities. The otsA gene has been cloned and recombinant protein overexpressed using a pET-based system in E. coli BL21 cells. The recombinant protein (MW approximately 54.7 kDa) is active and has been crystallized in two forms suitable for X-ray diffraction analysis. The first is orthorhombic, P2(1)2(1)2(1), with unit-cell parameters a = 104.1, b = 127.8, c = 179.9 A. Data for this form have been collected to 3.0 A resolution at the CLRC Daresbury Synchrotron Radiation Source. The second form has unit-cell parameters a = b = 141.9, c = 317.8 A and displays the apparent space group P4(2). These crystals diffract beyond 2 A resolution, but display merohedral twinning.

Inhibition of Human α-Methylacyl CoA Racemase (AMACR): a Target for Prostate Cancer

The enzyme α‐methylacyl CoA racemase (AMACR) is involved in the metabolism of branched‐chain fatty acids and has been identified as a promising therapeutic target for prostate cancer. By using the recently available human AMACR from HEK293 kidney cell cultures, we tested a series of new rationally designed inhibitors to determine the structural requirements in the acyl component. An N‐methylthiocarbamate (Ki=98 nM), designed to mimic the proposed enzyme‐bound enolate, was found to be the most potent AMACR inhibitor reported to date.

Trifluoroibuprofen Inhibits α-Methylacyl Coenzyme A Racemase (AMACR/P504S), Reduces Cancer Cell Proliferation and Inhibits in vivo Tumor Growth in Aggressive Prostate Cancer Models

BACKGROUND α-Methylacyl-CoA racemase (AMACR) participates in the oxidation of branched chain fatty acids and is highly expressed in prostate cancer (PCa). The aims of this study were to verify if the AMACR inhibitor trifluoroibuprofen (TFIP) had anticancer effects and to determine the best route for in vivo administration. MATERIALS AND METHODS In vitro effects of TFIP were verified by using three non-tumour prostate epithelial cell lines, a series of eight PCa cell lines and six cell derivatives. In vivo experiments were performed using PC3 and 22rv1 xenografts grown in nude mice with TFIP administered intraperitoneally or by oral gavage. RESULTS AMACR was expressed in PCa cell lines but was absent in normal and BPH cells. Although androgen-independent (AI) cell lines originating from androgen-dependent (AD) LnCaP cells displayed increased AMACR expression, the levels of this enzyme were higher in AI with respect to AD cell lines. TFIP induced: (1) down-modulation of AMACR expression; (2) suppression of the survival Akt/mTOR signalling pathway and (3) down-modulation of cyclin D1 and survivin with G2/M arrest and apoptosis. TFIP exhibited antitumour effects independently of the administration method. Nevertheless, oral administration was associated with acute toxicity at doses >75 mg/Kg/day. A dose of 75 mg/Kg administered biweekly reduced the toxicity whereas limited toxic effects were observed at 50 mg/Kg/day. Intraperitoneal administration of 75-100 mg/Kg/day was not toxic. CONCLUSIONS AMACR is a good pharmacological target for treatment of PCa and TFIP is a suitable anticancer compound with parenteral administration being the preferred route.

The oxygenating constituent of 3,6-diketocamphane monooxygenase from the CAM plasmid of Pseudomonas putida: the first crystal structure of a type II Baeyer–Villiger monooxygenase

The first crystal structure of a type II Baeyer–Villiger monooxygenase reveals a different ring orientation of its FMN cofactor compared with other related bacterial luciferase-family enzymes.

Expression and purification of the aortic amyloid polypeptide medin

The 50-amino acid protein medin is the main fibrillar component of human aortic medial amyloid (AMA), the most common form of localised amyloid which affects 97% of Caucasians over the age of 50. Structural models for several amyloid assemblies, including the Alzheimers amyloid-β peptides, have been defined from solid-state nuclear magnetic resonance (SSNMR) measurements on (13)C- and (15)N-labelled protein fibrils. SSNMR-derived structural information on fibrillar medin is scant, however, because studies to date have been restricted to limited measurements on site-specifically labelled protein prepared by solid-phase synthesis. Here we report a procedure for the expression of a SUMO-medin fusion protein in Escherichia coli and IMAC purification yielding pure, uniformly (13)C,(15)N-labelled medin in quantities required for SSNMR analysis. Thioflavin T fluorescence and dynamic light scattering measurements and transmission electron microscopy analysis confirm that recombinant medin assembles into amyloid-like fibrils over a 48-h period. The first (13)C and (15)N SSNMR spectra obtained for uniformly-labelled fibrils indicate that medin adopts a predominantly β-sheet conformation with some unstructured elements, and provide the basis for further, more detailed structural investigations.