Plants genetically modified by the pathogenic Agrobacterium strain C58 synthesize agrocinopines A and B, whereas those modified by the pathogenic strain Bo542 produce agrocinopines C and D. The four agrocinopines (A, B, C and D) serve as nutrients by agrobacteria and signaling molecule for the dissemination of virulence genes. They share the uncommon pyranose-2-phosphate motif, represented by the l-arabinopyranose moiety in agrocinopines A/B and the d-glucopyranose moiety in agrocinopines C/D, also found in the antibiotic agrocin 84. They are imported into agrobacterial cytoplasm via the Acc transport system, including the solute-binding protein AccA coupled to an ABC transporter. We have previously shown that unexpectedly, AccA from strain C58 (AccAC58) recognizes the pyranose-2-phosphate motif present in all four agrocinopines and agrocin 84, meaning that strain C58 is able to import agrocinopines C/D, originating from the competitor strain Bo542. Here, using agrocinopine derivatives and combining crystallography, affinity and stability measurements, modeling, molecular dynamics, in vitro and vivo assays, we show that AccABo542 and AccAC58 behave differently despite 75% sequence identity and a nearly identical ligand binding site. Indeed, strain Bo542 imports only compounds containing the d-glucopyranose-2-phosphate moiety, and with a lower affinity compared with strain C58. This difference in import efficiency makes C58 more competitive than Bo542 in culture media. We can now explain why Agrobacterium/Allorhizobium vitis strain S4 is insensitive to agrocin 84, although its genome contains a conserved Acc transport system. Overall, our work highlights AccA proteins as a case study, for which stability and dynamics drive specificity.

Supplementary Data from The Effect of Ketoconazole on the Pharmacokinetics and Pharmacodynamics of Ixabepilone: A First in Class Epothilone B Analogue in Late-Phase Clinical Development

Increased metabolism is one of the main causes for evolution of herbicide resistance in weeds, a major challenge for sustainable food production. The molecular drivers of this evolution are poorly understood. We tested here the hypothesis that a suitable context for the emergence of herbicide resistance could be provided by plant enzymes with high innate promiscuity with regard to their natural substrates. A selection of yeast-expressed plant cytochrome P450 enzymes with well documented narrow to broad promiscuity when metabolizing natural substrates was tested for herbicide metabolism competence. The positive candidate was assayed for capacity to confer herbicide tolerance in Arabidopsis thaliana. Our data demonstrate that Arabidopsis thaliana CYP706A3, with the most promiscuous activity on monoterpenes and sesquiterpenes for flower defence, can also oxidize plant microtubule assembly inhibitors, dinitroanilines. Ectopic overexpression of CYP706A3 confers dinitroaniline resistance. We show, in addition, that the capacity to metabolize dinitroanilines is shared by other members of the CYP706 family from plants as diverse as eucalyptus and cedar. Supported by three-dimensional (3D) modelling of CYP706A3, the properties of enzyme active site and substrate access channel are discussed together with the shared physicochemical properties of the natural and exogenous substrates to explain herbicide metabolism.

Ivermectin Toxicity and Nonsense Mutations Encephalopathy and coma developed in a 13-year-old boy shortly after he received a single dose of ivermectin to prevent scabies infection. ABCB1 sequencin...

Prevention therapy against Dirofilaria immitis in companion animals is currently threatened by the emergence of isolates resistant to macrocyclic lactone anthelmintics. Understanding the control over developmental processes in D. immitis is important for elucidating new approaches to heartworm control. The nuclear receptor DAF-12 plays a role in the entry and exit of dauer stage in Caenorhabditis elegans and in the development of free-living infective third-stage larvae (iL3) of some Clade IV and V parasitic nematodes. We identified a DAF-12 ortholog in the clade III nematode D. immitis and found that it exhibited a much higher affinity for dafachronic acids than described with other nematode DAF-12 investigated so far. We also modelled the DimDAF-12 structure and characterized the residues involved with DA binding. Moreover, we showed that cholesterol derivatives impacted the molting process from the iL3 to the fourth-stage larvae . Since D. immitis is unable to synthesize cholesterol and only completes its development upon host infection, we hypothesize that host environment contributes to its further molting inside the host vertebrate. Our discovery contributes to a better understanding of the developmental checkpoints of D. immitis and offers new perspectives for the development of novel therapies against filarial infections.

Ivermectin Toxicity and Nonsense Mutations Encephalopathy and coma developed in a 13-year-old boy shortly after he received a single dose of ivermectin to prevent scabies infection. ABCB1 sequencin...

We computed the network of channels of the 3A4 isoform of the cytochrome P450 (CYP) on the basis of 16 crystal structures extracted from the Protein Data Bank (PDB). The calculations were performed with version 2 of the CCCPP software that we developed for this research project. We identified the minimal cost paths (MCPs) output by CCCPP as probable ways to access to the buried active site. The algorithm of calculation of the MCPs is presented in this paper, with its original method of visualization of the channels. We found that these MCPs constitute four major channels in CYP3A4. Among the many channels proposed by Cojocaru et al. in 2007, we found that only four of them open in 3A4. We provide a refined description of these channels together with associated quantitative data.

Introduction and purpose of the study: Vitamin D is an essential molecule for phosphocalcic homeostasis. It would also play a key role in the prevention of a number of immune and metabolic pathologies, as well as with respect to certain cancers. However, vitamin D deficiencies and sub-deficiencies remain a major public health problem in both developing and industrialized countries. Better understanding the intestinal transport of vitamin D could optimize the status of individuals. We therefore investigated the involvement of ATP binding cassette B1 (ABCB1) in the intestinal efflux of vitamin D. Material and methods: To carry out this study, we have implemented a translational approach of the protein to humans by combining in silico, in vitro, ex vivo and in vivo models in mice - especially in mice deficient in ABCB1 ( Abcb1 - / - ), and clinical. These models allowed us to evaluate the efflux of neo-absorbed vitamin D, but also the transfer of vitamin D from the plasma to the intestinal lumen. Results and statistical analysis: Apical efflux of cholecalciferol (dietary vitamin D) and 25-hydroxycholecalciferol (circulating vitamin D) were decreased by chemical inhibition in Caco-2 TC7 cells (up to -34.9%, p < 0.05), and increased by overexpression of ABCB1 in Griptites or MDCKII cells (up to + 167.1%, p < 0.05). The Abcb1 - / - mice had a significant accumulation of 25-hydroxycholecalciferol in the plasma (42.6%), intestines (+ 43.0%), brain (+ 58.2%), liver ( +59, 2%) and kidneys (+ 20.6%), as well as a postprandial cholecalciferol response increased after gavage 3.43 and 6.71 μmol.h / g of lipids for Abcb1 - / - mice and controls, respectively. ( p < 0.05). The efflux of 25-hydroxycholecalciferol by intestinal explants from Abcb1 - / - mice was halved compared to control explants ( p < 0.05). This reduction in the transfer of 25-hydroxycholecalciferol from the plasma to the intestinal lumen was confirmed in vivo in mice perfused in situ at the intestinal level (-36.6% in the transgenic mice compared to the controls, p < 0.05). In silico experiments have shown that both cholecalciferol and 25-hydroxycholecalciferol can bind with high affinity to the P-glycoprotein of Caenorhabditis elegans, used as model ABCB1. Finally, in a group of 39 healthy adult men, an SNP in ABCB1 (rs17 064) was significantly associated with fasting plasma concentration of 25-hydroxycholecalciferol (35.94 ± 4.50 nmol / L, n = 9 vs 56.07 ± 3.00 nmol / L, n = 30, adjusted p = 0.041, t-test with Benjamini-Hochberg correction). Conclusion: We have shown for the first time that ABCB1 is involved in the efflux of vitamin D neo-absorbed by enterocytes, and that ABCB1 also contributes to the transintestinal excretion of vitamin D.

Flowers are essential but vulnerable plant organs, exposed to pollinators and florivores; however, flower chemical defenses are rarely investigated. We show here that two clustered terpene synthase and cytochrome P450 encoding genes (TPS11 and CYP706A3) on chromosome 5 of Arabidopsis (Arabidopsis thaliana) are tightly coexpressed in floral tissues, upon anthesis and during floral bud development. TPS11 was previously reported to generate a blend of sesquiterpenes. By heterologous coexpression of TPS11 and CYP706A3 in yeast (Saccharomyces cerevisiae) and Nicotiana benthamiana, we demonstrate that CYP706A3 is active on TPS11 products and also further oxidizes its own primary oxidation products. Analysis of headspace and soluble metabolites in cyp706a3 and 35S:CYP706A3 mutants indicate that CYP706A3-mediated metabolism largely suppresses sesquiterpene and most monoterpene emissions from opening flowers, and generates terpene oxides that are retained in floral tissues. In flower buds, the combined expression of TPS11 and CYP706A3 also suppresses volatile emissions and generates soluble sesquiterpene oxides. Florivory assays with the Brassicaceae specialist Plutella xylostella demonstrate that insect larvae avoid feeding on buds expressing CYP706A3 and accumulating terpene oxides. Composition of the floral microbiome appears also to be modulated by CYP706A3 expression. TPS11 and CYP706A3 simultaneously evolved within Brassicaceae and form the most versatile functional gene cluster described in higher plants so far.

Expérimental/mécanismes cellulaires et moléculaires. La vitamine D est une molécule essentielle à l’homéostasie phosphocalcique. Elle jouerait également un rôle clé dans la prévention d’un certain nombre de pathologies immunitaires et métaboliques, ainsi que vis-à-vis de certains cancers. Pourtant, les carences et les subcarences en vitamine D restent un problème majeur de santé public, aussi bien dans les pays en voie de développement que dans les pays industrialisés. Mieux comprendre le transport intestinal de la vitamine D pourrait permettre d’optimiser le statut des individus. Nous avons donc étudié l’implication de l’ATP binding cassette B1 (ABCB1) dans l’efflux intestinal de vitamine D. Pour mener à bien cette étude, nous avons mis en place une approche translationnelle de la protéine à l’homme en combinant des modèles in silico, in vitro, ex vivo et in vivo chez la souris - notamment chez des souris déficientes en ABCB1 (Abcb1 -/-), et clinique. Ces modèles nous ont permis d’évaluer l’efflux de vitamine D néo-absorbée, mais aussi le transfert de la vitamine D du plasma vers la lumière intestinale. Les efflux apicaux de cholécalciférol (vitamine D alimentaire) et de 25-hydroxycholécalciférol (vitamine D circulante) étaient diminués par inhibition chimique dans les cellules Caco-2 TC7 (jusqu’à -34,9 %, p < 0,05), et augmentés par la surexpression d’ABCB1 dans les Griptites ou les cellules MDCKII (jusqu’à + 167,1 %, p < 0,05). Les souris Abcb1 -/- présentaient une accumulation significative de 25-hydroxycholécalciférol dans le plasma (42,6 %), les intestins (+ 43,0 %), le cerveau (+ 58,2 %), le foie (+ 59,2 %) et les reins (+ 20,6 %), ainsi qu’une réponse postprandiale en cholécalciférol accrue après gavage 3,43 and 6,71 μmol.h/g de lipides pour les souris Abcb1-/- et témoins, respectivement (p < 0,05). L’efflux de 25-hydroxycholécalciférol par des explants intestinaux issus de souris Abcb1 -/- étaient réduit de moitié par rapport aux explants témoins (p < 0,05). Cette réduction du transfert de 25-hydroxycholécalciférol du plasma à la lumière intestinale a été confirmée in vivo chez des souris perfusées in situ au niveau intestinale (-36,6 % chez les souris transgéniques par rapport au témoins, p < 0,05). Des expériences in silico ont permis d’établir que le cholécalciférol et le 25-hydroxycholécalciférol pouvaient tous deux se lier avec une grande affinité à la P-glycoprotéine de Caenorhabditis elegans, utilisée comme modèle ABCB1. Enfin, dans un groupe de 39 hommes adultes en bonne santé, un SNP dans ABCB1 (rs17 064) était significativement associé à la concentration plasmatique en 25-hydroxycholécalciférol à jeun (35,94 ± 4,50 nmol/L, n = 9 vs 56,07 ± 3,00 nmol/L, n = 30 ; p ajustée = 0,041, t-test avec correction de Benjamini-Hochberg). Nous avons ainsi montré pour la première fois qu’ABCB1 est impliqué dans l’efflux de vitamine D néo-absorbés par les entérocytes, et qu’ABCB1 contribue également à l’excrétion transintestinale de vitamine D.

During the evolution of cellular bioenergetics, many protein families have been fashioned to match the availability and replenishment in energy supply. Molecular motors and primary transporters essentially need ATP to function while proteins involved in cell signaling or translation consume GTP. ATP-Binding Cassette (ABC) transporters are one of the largest families of membrane proteins gathering several medically relevant members that are typically powered by ATP hydrolysis. Here, a Streptococcus pneumoniae ABC transporter responsible for fluoroquinolones resistance in clinical settings, PatA/PatB, is shown to challenge this concept. It clearly favors GTP as the energy supply to expel drugs. This preference is correlated to its ability to hydrolyze GTP more efficiently than ATP, as found with PatA/PatB reconstituted in proteoliposomes or nanodiscs. Importantly, the ATP and GTP concentrations are similar in S. pneumoniae supporting the physiological relevance of GTP as the energy source of this bacterial transporter.

The influence of Arginine 117 of human cytochrome P450 2J2 in the recognition of ebastine and a series of terfenadone derivatives was studied by site-directed mutagenesis. R117K, R117E, and R117L mutants were produced, and the behavior of these mutants in the hydroxylation of ebastine and terfenadone derivatives was compared to that of wild-type CYP2J2. The data clearly showed the importance of the formation of a hydrogen bond between R117 and the keto group of these substrates. The data were interpreted on the basis of 3D homology models of the mutants and of dynamic docking of the substrates in their active site. These modeling studies also suggested the existence of a R117-E222 salt bridge between helices B’ and F that would be important for maintaining the overall folding of CYP2J2.

Efficient intestinal absorption of dietary vitamin D is required in most people to ensure an adequate status. Thus, we investigated the involvement of ATP binding cassette subfamily B member 1 (ABCB1) in vitamin D intestinal efflux. Both cholecalciferol (D3) and 25‐hydroxycholecalciferol [25(OH)D3] apical effluxes were decreased by chemical inhibition of ABCB1 in Caco‐2 cells and increased by ABCB1 overexpression in Griptites or Madin‐Darby canine kidney type II cells. Mice deficient for the 2 murine ABCB1s encoded by Abcb1a and Abcb1b genes (Abcb1−/−) displayed an accumulation of 25(OH)D3 in plasma, intestine, brain, liver, and kidneys, together with an increased D3 postprandial response after gavage compared with controls. 25(OH)D3 efflux through Abcb1−/− intestinal expiants was markedly decreased compared with controls. This reduction of 25(OH)D3 transfer from plasma to lumen was further confirmed in vivo in intestine‐perfused mice. Docking experiments established that both D3 and 25(OH)D3 could bind with high affinity to Caenorhabditis elegans P‐glycoprotein, used as an ABCB1 model. Finally, in a group of 39 healthy male adults, a single‐nucleotide polymorphism (SNP) in ABCB1 (rs17064) was significantly associated with the fasting plasma 25(OH)D3 concentration. Thus, we showed here for the first time that ABCB1 is involved in neo‐absorbed vitamin D efflux by the enterocytes and that it also contributes to vitamin D transintestinal excretion and likely impacts vitamin D status.—Margier, M., Collet, X., leMay, C., Desmarchelier, C., André, F., Lebrun, C., Defoort, C., Bluteau, A., Borel, P., Lespine, A., Reboul, E. ABCB1 (P‐glycoprotein) regulates vitamin D absorption and contributes to its transintestinal efflux. FASEB J. 33, 2084–2094 (2019). www.fasebj.org

Haemonchus contortus, one of the most economically important parasites of small ruminants, has become resistant to the anthelmintic ivermectin. Deciphering the role of P-glycoproteins in ivermectin resistance is desirable for understanding and overcoming this resistance. In the model nematode, Caenorhabditis elegans, P-glycoprotein-13 is expressed in the amphids, important neuronal structures for ivermectin activity. We have focused on its ortholog in the parasite, Hco-Pgp-13. A 3D model of Hco-Pgp-13, presenting an open inward-facing conformation, has been constructed by homology with the Cel-Pgp-1 crystal structure. In silico docking calculations predicted high affinity binding of ivermectin and actinomycin D to the inner chamber of the protein. Following in vitro expression, we showed that ivermectin and actinomycin D modulated Hco-Pgp-13 ATPase activity with high affinity. Finally, we found in vivo Hco-Pgp-13 localization in epithelial, pharyngeal and neuronal tissues. Taken together, these data suggest a role for Hco-Pgp-13 in ivermectin transport, which could contribute to anthelmintic resistance.

Background: Cytochrome P450 2U1 (CYP2U1) has been identified from the human genome and is highly conserved in the living kingdom. It is considered as an "orphan" protein as few data are available on its physiological function(s) and spectral characteristics. Its only known substrates reported so far are unsaturated fatty acids such as arachidonic acid (AA), and, more recently, N-arachidonoylserotonin (AS) and some xenobiotics related to debrisoquine (Deb) and terfenadine. Methods: We have expressed CYP2U1 in E. coli and performed UV-vis and EPR spectroscopy experiments with purified CYP2U1 alone and in the presence of substrates and imidazole and pyridine derivatives. Docking experiments using a 3D homology model of CYP2U1 were done to explain the observed spectroscopic data and the different regioselectivities of the oxidations of AA and AS. Results: The UV-vis and EPR spectra of native recombinant human CYP2U1 revealed a predominant low-spin hexacoordinate Fe-III state. Imidazole (Im) derivatives, such as miconazole, acted as Fe-III ligands, contrary to ketoconazole, whereas the previously described substrates AS and Deb led to "reverse type I" difference UV-vis spectra. These data, as well as the different regioselectivities of AA and AS oxidations, were supported by docking experiments performed on our previously reported CYP2U1 3D model. Major conclusion and general significance: Our study describes for the first time the mode of interaction of several Fe-III-heme ligands and substrates with the active site of CYP2U1 on the basis of spectroscopic and molecular docking data. The good agreement between these data validates the used CYP2U1 3D model which should help the design of new substrates or inhibitors of this orphan CYP. (C) 2016 Elsevier B.V. All rights reserved.

Evolution of the phenolic metabolism was critical for the transition of plants from water to land. A cytochrome P450, CYP73, with cinnamate 4-hydroxylase (C4H) activity, catalyzes the first plant-specific and rate-limiting step in this pathway. The CYP73 gene is absent from green algae, and first detected in bryophytes. A CYP73 duplication occurred in the ancestor of seed plants and was retained in Taxaceae and most angiosperms. In spite of a clear divergence in primary sequence, both paralogs can fulfill comparable cinnamate hydroxylase roles both in vitro and in vivo. One of them seems dedicated to the biosynthesis of lignin precursors. Its N-terminus forms a single membrane spanning helix and its properties and length are highly constrained. The second is characterized by an elongated and variable N-terminus, reminiscent of ancestral CYP73s. Using as proxies the Brachypodium distachyon proteins, we show that the elongation of the N-terminus does not result in an altered subcellular localization, but in a distinct membrane topology. Insertion in the membrane of endoplasmic reticulum via a double-spanning open hairpin structure allows reorientation to the lumen of the catalytic domain of the protein. In agreement with participation to a different functional unit and supramolecular organization, the protein displays modified heme proximal surface. These data suggest the evolution of divergent C4H enzymes feeding different branches of the phenolic network in seed plants. It shows that specialization required for retention of gene duplicates may result from altered protein topology rather than change in enzyme activity.

The exponential increase of genomes' sequencing has revealed the presence of NO-Synthases (NOS) throughout the tree of life, uncovering an extraordinary diversity of genetic structure and biological functions. Although NO has been shown to be a crucial mediator in plant physiology, NOS sequences seem present solely in green algae genomes, with a first identification in the picoplankton species Ostreococcus tauri. There is no rationale so far to account for the presence of NOS in this early-diverging branch of the green lineage and its absence in land plants. To address the biological function of algae NOS, we cloned, expressed and characterized the NOS oxygenase domain from Ostreococcus tauri (OtNOSoxy). We launched a phylogenetic and structural analysis of algae NOS, and achieved a 3D model of OtNOSoxy by homology modeling. We used a combination of various spectroscopies to characterize the structural and electronic fingerprints of some OtNOSoxy reaction intermediates. The analysis of OtNOSoxy catalytic activity and kinetic efficiency was achieved by stoichiometric stopped-flow. Our results highlight the conserved and particular features of OtNOSoxy structure that might explain its ultrafast NO-producing capacity. This integrative Structure-Catalysis-Function approach could be extended to the whole NOS superfamily and used for predicting potential biological activity for any new NOS.

Over the past twenty years, nitric oxide (NO) has emerged as an important player in various plant physiological processes. Although many advances in the understanding of NO functions have been made, the question of how NO is produced in plants is still challenging. It is now generally accepted that the endogenous production of NO is mainly accomplished through the reduction of nitrite via both enzymatic and non-enzymatic mechanisms which remain to be fully characterized. Furthermore, experimental arguments in favour of the existence of plant nitric oxide synthase (NOS)-like enzymes have been reported. However, recent investigations revealed that land plants do not possess animal NOS-like enzymes while few algal species do. Phylogenetic and structural analyses reveals interesting features specific to algal NOS-like proteins.

Background: Human cytochrome P450 2U1 (CYP2U1) is an orphan CYP that exhibits several distinctive characteristics among the 57 human CYPs with a highly conserved sequence in almost all living organisms.Methods: We compared its protein sequence with those of the 57 human CYPs and constructed a 3D structure of a full-length CYP2U1 model bound to a POPC membrane. We also performed docking experiments of arachidonic acid (AA) and N-arachidonoylserotonin (AS) in this model.Results: The protein sequence of CYP2U1 displayed two unique characteristics when compared to those of the human CYPs, the presence of a longer N-terminal region upstream of the putative trans-membrane helix (TMH) containing 8 proline residues, and of an insert of about 20 amino acids containing 5 arginine residues between helices A' and A. Its N-terminal part upstream of TMH involved an additional short terminal helix, in a manner similar to what was reported in the crystal structure of Saccharomyces cerevisiae CYP51. Our model also showed a specific interaction between the charged residues of insert AA' and phosphate groups of lipid polar heads, suggesting a possible role of this insert in substrate recruitment. Docking of AA and AS in this model showed these substrates in channel 2ac, with the terminal alkyl chain of AA or the indole ring of AS close to the heme, in agreement with the reported CYP2U1-catalyzed AA and AS hydroxylation regioselectivities.Major conclusion and general significance: This model should be useful to find new endogenous or exogenous CYP2U1 substrates and to interpret the regioselectivity of their hydroxylation. (C) 2017 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.

We computed the channels of the 3A4 isoform of the cytochrome P450 3A4 (CYP) on the basis of 24 crystal structures extracted from the Protein Data Bank (PDB). We identified three major conformations (denoted C, O1 and O2) using an enhanced version of the CCCPP software that we developed for the present work, while only two conformations (C and O2 ) are considered in the literature. We established the flowchart of definition of these three conformations in function of the structural and physicochemical parameters of the ligand. The channels are characterized with qualitative and quantitative parameters, and not only with their surrounding secondary structures as it is usually done in the literature.