François André

Geraniol hydroxylase and hydroxygeraniol oxidase activities of the CYP76 family of cytochrome P450 enzymes and potential for engineering the early steps of the (seco)iridoid pathway.

The geraniol-derived (seco)iridoid skeleton is a precursor for a large group of bioactive compounds with diverse therapeutic applications, including the widely used anticancer molecule vinblastine. Despite of this economic prospect, the pathway leading to iridoid biosynthesis from geraniol is still unclear. The first geraniol hydroxylation step has been reported to be catalyzed by cytochrome P450 enzymes such as CYP76B6 from Catharanthus roseus and CYP76C1 from Arabidopsis thaliana. In the present study, an extended functional analysis of CYP76 family members was carried-out to identify the most effective enzyme to be used for pathway reconstruction. This disproved CYP76C1 activity and led to the characterization of CYP76C4 from A. thaliana as a geraniol 9- or 8-hydroxylase. CYP76B6 emerged as a highly specialized multifunctional enzyme catalyzing two sequential oxidation steps leading to the formation of 8-oxogeraniol from geraniol. This dual function was confirmed in planta using a leaf-disc assay. The first step, geraniol hydroxylation, was very efficient and fast enough to outcompete geraniol conjugation in plant tissues. When the enzyme was expressed in leaf tissues, 8-oxogeraniol was converted into further oxidized and/or reduced compounds in the absence of the next enzyme of the iridoid pathway.

Nitric oxide synthase in plants: Where do we stand?

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.

Evolutionary interplay between sister cytochrome P450 genes shapes plasticity in plant metabolism

Expansion of the cytochrome P450 gene family is often proposed to have a critical role in the evolution of metabolic complexity, in particular in microorganisms, insects and plants. However, the molecular mechanisms underlying the evolution of this complexity are poorly understood. Here we describe the evolutionary history of a plant P450 retrogene, which emerged and underwent fixation in the common ancestor of Brassicales, before undergoing tandem duplication in the ancestor of Brassicaceae. Duplication leads first to gain of dual functions in one of the copies. Both sister genes are retained through subsequent speciation but eventually return to a single copy in two of three diverging lineages. In the lineage in which both copies are maintained, the ancestral functions are split between paralogs and a novel function arises in the copy under relaxed selection. Our work illustrates how retrotransposition and gene duplication can favour the emergence of novel metabolic functions.

In silico analysis of the binding of anthelmintics to Caenorhabditis elegansP-glycoprotein 1

Macrocyclic lactones (ML) are important anthelmintics used in animals and humans against parasite nematodes, but their therapeutic success is compromised by the spread of ML resistance. Some ABC transporters, such as p-glycoproteins (Pgps), are selected and overexpressed in ML-resistant nematodes, supporting a role for some drug efflux proteins in ML resistance. However, the role of such proteins in ML transport remains to be clarified at the molecular level. Recently, Caenorhabditis elegans Pgp-1 (Cel-Pgp-1) has been crystallized, and its drug-modulated ATPase function characterized in vitro revealed Cel-Pgp-1 as a multidrug transporter. Using this crystal structure, we have developed an in silico drug docking model in order to study the binding of ML and other anthelmintic drugs to Cel-Pgp-1. All tested ML bound with high affinity in a unique site, within the inner chamber of the protein, supporting that ML may be transported by Cel-Pgp-1. Interestingly, interacting residues delineate a ML specific fingerprint involving H-bonds, including T1028. In particular, benzofurane and spiroketal moieties bound to specific sub-sites. When compared with the aglycone ML, such as moxidectin and ivermectin aglycone, avermectin anthelmintics have significant higher affinity for Cel-Pgp-1, likely due to the sugar substituent(s) that bind to a specific area involving H-bonds at Y771. Triclabendazole, closantel and emodepside bound with good affinities to different sub-sites in the inner chamber, partially overlapping with the ML binding site, suggesting that they could compete for Cel-Pgp-1-mediated ML transport. In conclusion, this work provides novel information on the role of nematode Pgps in transporting anthelmintics, and a valuable tool to predict drug-drug interactions and to rationally design new competitive inhibitors of clinically-relevant nematode Pgps, to improve anthelmintic therapeutics.

Gene Duplication Leads to Altered Membrane Topology of a Cytochrome P450 Enzyme in Seed Plants

Abstract 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 NOS-like protein from the microalgae Ostreococcus tauri is a genuine and ultrafast NO-producing enzyme

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.

The Cytochrome P450 3A4 has three Major Conformations: New Clues to Drug Recognition by this Promiscuous Enzyme

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

Lactococcus lactis is an Efficient Expression System for Mammalian Membrane Proteins Involved in Liver Detoxification, CYP3A4, and MGST1

Despite the great importance of human membrane proteins involved in detoxification mechanisms, their wide use for biochemical approaches is still hampered by several technical difficulties considering eukaryotic protein expression in order to obtain the large amounts of protein required for functional and/or structural studies. Lactococcus lactis has emerged recently as an alternative heterologous expression system to Escherichia coli for proteins that are difficult to express. The aim of this work was to check its ability to express mammalian membrane proteins involved in liver detoxification, i.e., CYP3A4 and two isoforms of MGST1 (rat and human). Genes were cloned using two different strategies, i.e., classical or Gateway-compatible cloning, and we checked the possible influence of two affinity tags (6×-His-tag and Strep-tag II). Interestingly, all proteins could be successfully expressed in L. lactis at higher yields than those previously obtained for these proteins with classical expression systems (E. coli, Saccharomyces cerevisiae) or those of other eukaryotic membrane proteins expressed in L. lactis. In addition, rMGST1 was fairly active after expression in L. lactis. This study highlights L. lactis as an attractive system for efficient expression of mammalian detoxification membrane proteins at levels compatible with further functional and structural studies.

Membrane Protein Production in Lactococcus lactis for Functional Studies.

Due to their unique properties, expression and study of membrane proteins in heterologous systems remains difficult. Among the bacterial systems available, the Gram-positive lactic bacterium, Lactococcus lactis, traditionally used in food fermentations, is nowadays widely used for large-scale production and functional characterization of bacterial and eukaryotic membrane proteins. The aim of this chapter is to describe the different possibilities for the functional characterization of peripheral or intrinsic membrane proteins expressed in Lactococcus lactis.

A multidrug ABC transporter with a taste for GTP

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.