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Cytochrome P450 3A4 (CYP3A4) is the most important drug-metabolizing enzyme in humans and has been associated with harmful drug interactions. The activity of CYP3A4 is known to be modulated by several compounds and by the electron transfer partner, cytochrome P450 reductase (CPR). The underlying mechanism of these effects, however, is poorly understood. We have used hydrogen-deuterium exchange mass spectrometry to investigate the impact of binding of CPR and of three different substrates (7-benzyloxy-4-trifluoromethyl-coumarin, testosterone, and progesterone) on the conformational dynamics of CYP3A4. Here, we report that interaction of CYP3A4 with substrates or with the oxidized or reduced forms of CPR leads to a global rigidification of the CYP3A4 structure. This was evident from the suppression of deuterium exchange in several regions of CYP3A4, including regions known to be involved in protein-protein interactions (helix C) and substrate binding and specificity (helices B and E, and loop K/β1). Furthermore, the bimodal isotopic distributions observed for some CYP3A4-derived peptides were drastically impacted upon binding to CPR and/or substrates, suggesting the existence of stable CYP3A4 conformational populations that are perturbed by ligand/CPR binding. The results have implications for understanding the mechanisms of ligand binding, allostery, and catalysis in CYP enzymes.

Itaconate is a conjugated 1,4-dicarboxylate produced by macrophages. This small molecule has recently received increasing attention due to its role in modulating the immune response of macrophages upon exposure to pathogens. Itaconate has also been proposed to play an antimicrobial function; however, this has not been explored as intensively. Consistent with the latter, itaconate is known to show antibacterial activity in vitro and was reported to inhibit isocitrate lyase, an enzyme required for survival of bacterial pathogens in mammalian systems. Recent studies have revealed bacterial growth inhibition under biologically relevant conditions. In addition, an antimicrobial role for itaconate is substantiated by the high concentration of itaconate found in bacteria-containing vacuoles, and by the production of itaconate-degrading enzymes in pathogens such as Salmonella enterica ser. Typhimurium, Pseudomonas aeruginosa, and Yersinia pestis. This review describes the current state of literature in understanding the role of itaconate as an antimicrobial agent in host-pathogen interactions. Key words: antimicrobial, antimicrobial resistance, isocitrate lyase, itaconic acid, glyoxylate cycle, macrophages. L'itaconate est un 1,4-dicarboxylate conjugué produit par les macrophages. Cette petite molécule suscite depuis peu un intérêt grandissant en raison de son rôle dans la modulation de la réponse immunitaire des macrophages face à une exposition à des agents pathogènes. Des chercheurs ont aussi avancé l'idée que l'itaconate jouerait un rôle antimicrobien; toutefois, cette hypothèse n'a pas été explorée de manière aussi intensive. On sait néanmoins que l'itaconate présente une activité antibactérienne in vitro et qu'il inhibe l'isocitrate lyase, une enzyme nécessaire à la survie des bactéries pathogènes chez les mammifères. Des études récentes ont révélé qu'il inhibe la croissance bactérienne dans des conditions pertinentes aux infections. De plus, la forte concentration d'itaconate trouvée dans les vacuoles qui contiennent les bactéries ainsi que la production d'enzymes dégradant l'itaconate par des agents pathogènes tels que Salmonella entérica ser. Typhimurium, Pseudomonas aeruginosa et Yersinia pestis viennent corroborer le rôle antimicrobien de l'itaconate. Cet article de synthèse décrit l'état actuel de la littérature et des connaissances quant au rôle de l'itaconate comme agent antimicrobien dans les interactions hôte-pathogène. [Traduit par la Rédaction] Mots-clés : antimicrobien, résistance aux antimicrobiens, isocitrate lyase, acide itaconique, cycle du glyoxylate, macrophages., Introduction Itaconate (or methylene succinate, Fig. 1, in red) was first isolated from fungi like Aspergillus sp. (1-2) Since its first isolation from natural sources, this conjugate dicarboxylate has been [...]

Human cytochrome P450 3A4 (CYP3A4) is an important drug metabolizing enzyme involved in a number of drug-drug and food-drug interactions. As such, much effort has been devoted into investigating its mechanism of interaction with ligands. CYP3A4 has one of the highest levels of substrate promiscuity for an enzyme, and can even bind multiple ligands simultaneously. The location and orientation of these ligands depend on the chemical structure and stoichiometry, and are generally poorly understood. In the case of the steroid testosterone, up to three copies of the molecule can associate with the enzyme at once, likely two in the active site and one at a postulated allosteric site. Recently, we demonstrated that steroid bioconjugation at the allosteric site results in an increase in activity of CYP3A4 toward testosterone and 7-benzyloxy-4-trifluoromethylcoumarin oxidation. Here, using the established bioconjugation methodology, we show how steroid bioconjugation at the allosteric site affects the heme spin state, the binding affinity (KS) of CYP3A4 for testosterone, as well as the enzyme coupling efficiency.

Waste polyester textiles are not recycled due to separation challenges and partial structural degradation during use and recycling. Chemical recycling of polyethylene terephthalate (PET) textiles through depolymerization can provide a feedstock of recycled monomers to make "as‐new" polymers. While enzymatic PET recycling is a more selective and more sustainable approach, methods in development, however, have thus far been limited to clean, high‐quality PET feedstocks, and require an energy‐intensive melt‐amorphization step ahead of enzymatic treatment. Here, high‐crystallinity PET in mixed PET/cotton textiles could be directly and selectively depolymerized to terephthalic acid (TPA) by using a commercial cutinase from Humicola insolens under moist‐solid reaction conditions, affording up to 30±2 % yield of TPA. The process was readily combined with cotton depolymerization through simultaneous or sequential application of the cellulase enzymes CTec2®, providing up to 83±4 % yield of glucose without any negative influence on the TPA yield. [ABSTRACT FROM AUTHOR]

A solution nuclear magnetic resonance (NMR) spectroscopic study of cyanide-inhibited, substrate-bound complex of uniformly (super 15)N-labeled human heme oxygenase (hHO) was performed for characterizing the active site with respect to the nature and identity of strong hydrogen bonds and the occupation of ordered water molecules within the hydrogen bonding network and an aromatic cluster on the distal side.

The roles of proximal thiolate iron ligand in cytochrome P450 were studied use resonance Raman, UV, circular dichroism and activity measurements.