Structural, Functional, and Spectroscopic Characterization of the Substrate Scope of the Novel Nitrating Cytochrome P450 TxtE

A novel cytochrome P450 enzyme, TxtE, was recently shown to catalyze the direct aromatic nitration of L-tryptophan. This unique chemistry inspired us to ask whether TxtE could serve as a platform for engineering new nitration biocatalysts to replace current harsh synthetic methods. As a first step toward this goal, and to better understand the wild-type enzyme, we obtained high-resolution structures of TxtE in its substrate-free and substrate-bound forms. We also screened a library of substrate analogues for spectroscopic indicators of binding and for production of nitrated products. From these results, we found that the wild-type enzyme accepts moderate decoration of the indole ring, but the amino acid moiety is crucial for binding and correct positioning of the substrate and therefore less amenable to modification. A nitrogen atom is essential for catalysis, and a carbonyl must be present to recruit the αB′1 helix of the protein to seal the binding pocket

The genetic interaction network of CCW12, a Saccharomyces cerevisiae gene required for cell wall integrity during budding and formation of mating projections

<p>Abstract</p> <p>Background</p> <p>Mannoproteins construct the outer cover of the fungal cell wall. The covalently linked cell wall protein Ccw12p is an abundant mannoprotein. It is considered as crucial structural cell wall component since in baker's yeast the lack of <it>CCW12 </it>results in severe cell wall damage and reduced mating efficiency.</p> <p>Results</p> <p>In order to explore the function of <it>CCW12</it>, we performed a Synthetic Genetic Analysis (SGA) and identified genes that are essential in the absence of <it>CCW12</it>. The resulting interaction network identified 21 genes involved in cell wall integrity, chitin synthesis, cell polarity, vesicular transport and endocytosis. Among those are <it>PFD1</it>, <it>WHI3</it>, <it>SRN2</it>, <it>PAC10</it>, <it>FEN1 </it>and <it>YDR417C</it>, which have not been related to cell wall integrity before. We correlated our results with genetic interaction networks of genes involved in glucan and chitin synthesis. A core of genes essential to maintain cell integrity in response to cell wall stress was identified. In addition, we performed a large-scale transcriptional analysis and compared the transcriptional changes observed in mutant <it>ccw12</it>Δ with transcriptomes from studies investigating responses to constitutive or acute cell wall damage. We identified a set of genes that are highly induced in the majority of the mutants/conditions and are directly related to the cell wall integrity pathway and cell wall compensatory responses. Among those are <it>BCK1</it>, <it>CHS3</it>, <it>EDE1</it>, <it>PFD1</it>, <it>SLT2 </it>and <it>SLA1 </it>that were also identified in the SGA. In contrast, a specific feature of mutant <it>ccw12</it>Δ is the transcriptional repression of genes involved in mating. Physiological experiments substantiate this finding. Further, we demonstrate that Ccw12p is present at the cell periphery and highly concentrated at the presumptive budding site, around the bud, at the septum and at the tip of the mating projection.</p> <p>Conclusions</p> <p>The combination of high throughput screenings, phenotypic analyses and localization studies provides new insight into the function of Ccw12p. A compensatory response, culminating in cell wall remodelling and transport/recycling pathways is required to buffer the loss of <it>CCW12</it>. Moreover, the enrichment of Ccw12p in bud, septum and mating projection is consistent with a role of Ccw12p in preserving cell wall integrity at sites of active growth.</p> <p>The microarray data produced in this analysis have been submitted to NCBI GEO database and GSE22649 record was assigned.</p

L'U(H)PLC et le transfert de méthodes HPLC/U(H)PLC

National audienc

Caractérisation des ADCs par LCxLC-UV/MS

National audienc

On-line comprehensive two-dimensional liquid chromatography as a tool for addressing industrial issues

International audienc

L’intérêt de la haute température en UHPLC

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Identification d’anomalies en HPLC/UHPLC

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2D-LC comprehensive vs 1D-LC. Comment maximiser les gains ?

National audienc

La LC x LC est-elle mature pour l’analyse de composés pharmaceutiques et biopharmaceutiques ?

National audienc

Étude et utilisation de la chromatographie en phase liquide à haute température

L'objectif de ce travail est l'étude théorique et la mise au point instrumentale d'une nouvelle technique chromatographique : la chromatographie en phase liquide à haute température (HTLC). Nous avons mis en oeuvre cette technique en optimisant les paramètres instrumentaux. Nous avons également montré à travers une étude thermodynamique et cinétique appuyée par de nombreux exemples, que cette technique permettait de diminuer d'un facteur 5 à 10 les temps d'analyse, d'améliorer la forme des pics et de réduire la teneur en modificateur organique dans la phase mobile. Le couplage avec le détecteur à ionisation de flamme (FID) après optimisation des paramètres de fonctionnement, a été réalisé avec des phases mobiles aqueuses. L'intérêt d'un tel couplage a été démontré par l'analyse de nombreuses familles de solutés non chromophores. La HTLC a également été couplée à la spectrométrie de masse et des applications variées ont permis de mettre en évidence l'avantage d'un tel couplageLYON1-BU.Sciences (692662101) / SudocSudocFranceF