Philippe Urban

Philippe Urban
French National Centre for Scientific Research | CNRS · Toulouse Biotchnology Institute

PhD
I am deeply invovlved in using AlphaFold2 possibilities to implement them in protein complex determination.

About

76
Publications
68,104
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2,779
Citations
Citations since 2017
17 Research Items
544 Citations
2017201820192020202120222023020406080100
2017201820192020202120222023020406080100
2017201820192020202120222023020406080100
2017201820192020202120222023020406080100
Introduction
I have studied Enzymology in Paris University at Orsay. I work at TBI in Toulouse (CNRS UMR5504) with a focus on the structural determinants of the substrate specificity with P450 enzymes (my "favorite" enzymes). I am also working on innovative projects in industrial biotechnologies at TWB (Toulouse White Biotechnology). My basic formation is both in enzymology: kinetics, protein structure-dynamics-activity relationships, and mechanisms of action. I am academic editor at Biomolecules.
Additional affiliations
January 2022 - January 2026
French Institute of Health and Medical Research
Position
  • Expert Evaluator at CSS1 Commission Scientifique Spécialisée : Cellular Molecular and Structural Biology
July 2013 - August 2013
Technical Institute of Cambodia, Phnom Penh
Position
  • Temporary Professor
Description
  • 15 hours of teaching in molecular biotechnology and enzymology. Funded by AUF (Agence Universitaire de la Francophonie)
June 2012 - July 2012
Technical Institute of Cambodia, Phnom-Penh
Position
  • Invited Professor
Description
  • Evaluation of the quality of the final exam procedure for engineer graduation.

Publications

Publications (76)
Article
The dehydroepiandrosterone (DHEA) 7alpha-hydroxylation in humans takes place in the liver, skin, and brain. These organs are targets for the glucocorticoid hormones where 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) activates cortisone through its reduction into cortisol. The putative interference of 7alpha-hydroxy-DHEA with the 11beta-...
Article
Full-text available
Yeast Saccharomyces cerevisiae offers a low-cost and efficient way to express heterologous P450s. Nevertheless, the limiting amounts of endogenous NADPH-cytochrome P450 reductase (CPR) present in this organism and the limited sequence similarity of yeast redox enzymes (CPR and cytochrome b5) with human liver or plant equivalents are severe limitati...
Article
Full-text available
Conformational dynamics plays a critical role for the function of multidomain electron transfer complexes. While crystallographic or NMR approaches allow detailed insight into structures, lower resolution methods like cryo-electron microscopy can provide more information on dynamics. In silico structure modelling using AlphaFold was recently succes...
Preprint
Full-text available
AlphaFold2 has transformed the way 3D structures of protein may be modeled, it allows an accuracy unprecedented, sometimes even better than the experimental structure. In this preprint, we have used some up-to-now undocumented properties of the Advanced version of AlpahFold2 in generating, due to variable instantiations, alternate structures of a m...
Article
Full-text available
The activity of microsomal cytochromes P450 (CYP) is strictly dependent on the supply of electrons provided by NADPH cytochrome P450 oxidoreductase (CPR). The variant nature of the isoform-specific proximal interface of microsomal CYPs implies that the interacting interface between the two proteins is degenerated. Recently, we demonstrated that spe...
Article
Full-text available
NADPH cytochrome P450 oxidoreductase (CPR) is the obligatory electron supplier that sustains the activity of microsomal cytochrome P450 (CYP) enzymes. The variant nature of the isoform-specific proximal interface of microsomal CYPs indicates that CPR is capable of multiple degenerated interactions with CYPs for electron transfer, through different...
Article
Full-text available
4-hydroxybenzoic acid (pHBA) is an important industrial precursor of muconic acid and liquid crystal polymers whose production is based on the petrochemical industry. In order to decrease our dependency on fossil fuels and improve sustainability, microbial engineering is a particularly appealing approach for replacing traditional chemical technique...
Article
Full-text available
NADPH-cytochrome P450 reductase (CPR) is the unique redox partner of microsomal cytochrome P450s (CYPs). CPR exists in a conformational equilibrium between open and closed conformations throughout its electron transfer (ET) function. Previously, we have shown that electrostatic and flexibility properties of the hinge segment of CPR are critical for...
Article
Full-text available
Cytochrome P450s (CYPs) are key enzymes involved in drug and xenobiotic metabolism. A wide array of in vitro methodologies, including recombinant sources, are currently been used to assess CYP catalysis, to identify the metabolic profile of compounds, potential drug-drug interactions, protein-protein interactions in the CYP enzyme complex and the r...
Article
Full-text available
Quantitative structure-activity relationships may bring invaluable information on structural elements of both enzymes and substrates that, together, govern substrate specificity. Buried active sites in cytochrome P450 enzymes are connected to the solvent by a network of channels exiting at the distal surface of the protein. This review presents dif...
Article
Full-text available
Quantitative structure-activity relationships may bring invaluable information on structural elements of both enzymes and substrates that, together, govern substrate specificity. Buried active sites in cytochrome P450 enzymes are connected to the solvent by a network of channels exiting at the distal surface of the protein. This review presents dif...
Article
Full-text available
NADPH-cytochrome P450 reductase (CPR) is a redox partner of microsomal cytochromes P450 and is a prototype of the diflavin reductase family. CPR contains 3 distinct functional domains: a FMN-binding domain (acceptor reduction), a linker (hinge), and a connecting/FAD domain (NADPH oxidation). It has been demonstrated that the mechanism of CPR exhibi...
Article
Full-text available
Glaucoma is a clinical entity with multifactorial etiology, a severe subtype occurs in infancy called primary congenital glaucoma (PCG). Three distinct levels interact sequentially to produce PCG: (i) genetic mutations mainly affecting the CYP1B1 gene, (ii) absence or dysregulation of a morphogen, and (iii) trabecular meshwork pathological changes...
Patent
Full-text available
The invention relates to a method for producing derivatives of O-α-glucosylated flavonoid, comprising at least one step of incubating a glucansucrase with a flavonoid and at least one sucrose, the flavonoid being a flavonoid which is monohydroxylated or hydroxylated in a non-vicinal manner on the B cycle. The invention also relates to novel O-α-glu...
Conference Paper
Full-text available
There are no apparent access pathways between the surface of a P450 protein and its catalytic cavity. It was shown by molecular dynamics simulations that P450 enzymes present channels for substrate access and product exit. CAVER algorithm finds a network of channels linking the catalytic cavity to the protein surface on the distal face of most P450...
Article
Full-text available
The promiscuity of a collection of enzymes consisting of 31 wild-type and synthetic variants of CYP1A enzymes was evaluated using a series of 14 steroids and 2 steroid-like chemicals, namely, nootkatone, a terpenoid, and mifepristone, a drug. For each enzyme-substrate couple, the initial steady-state velocity of metabolite formation was determined...
Data
Full-text available
Table S1. Table of specific activities observed with the different enzymes when assayed for the different steroidal substrates of this work (in five parts). EOR, MOR, EFEE stands for 7-ethoxy-, 7-methoxy-resorufin, and 7-ethoxy-fluorescein ethyl ester, respectively. The different hydroxylated metabolites observed in LC/MS are designated by one or t...
Conference Paper
Full-text available
Article
Interindividual variability in cytochrome P450 (CYP)-mediated xenobiotic metabolism is extensive. CYP metabolism requires two electrons, which can be donated by NADPH cytochrome P450 oxidoreductase (CYPOR) and/or cytochrome b5 (b5). Although substantial number of studies have reported on the function and effect of b5 in CYP-mediated catalysis, its...
Article
La biologie de synthèse vise à recomposer, en utilisant les briques du vivant, des systèmes variés de la molécule à l’organisme en suivant une logique qui peut s’éloigner des systèmes biologiques naturels pour créer de nouvelles fonctions d’intérêt. L’ingénierie métabolique a pris un nouvel essor dans ce cadre en mettant à profit les approches in s...
Article
Full-text available
The ability of four mammalian cytochromes P450 (CYP) of the CYP1A subfamily, human and mouse CYP1A1s and human and rabbit CYP1A2s, to metabolize a series of steroids and related compounds was investigated us-ing high throughput approaches. Oxidation rates and metabolite patterns for 16 steroid substrates and for 20 polycyclic aromatic hydrocarbon (...
Article
Full-text available
High-throughput (HT) characterization of drugs for potential biotransformation and interaction is routine in pharmaceutical industry. HT approaches were extended to enzyme studies for identifying combinations of structural elements that control substrate specificity. Structure-based and combinatorial mutagenesis have been applied with success to de...
Article
A comparison of all known mammalian CYP1A sequences identifies nineteen sequence regions that are conserved within all 1A1s or within all 1A2s but at the same time systematically differ between any 1A1 and any 1A2. The purpose of this study was to explore links between these specific CYP1A sequence signatures and substrate specificity shift through...
Article
Cytochromes P450 constitute a superfamily of haem-thiolate mono-oxygenases that are involved in the oxidative metabolism of lipophilic subtrates. These enzymes require association with cytochrome P450 reductase (CPR) to achieve optimal activities. We have expressed human cytochrome P450 CYP1A1 under the POX2 promoter (pPOX2-CYP1A1) in Y. lipolytica...
Article
Abstract Two complementary methods are described that associate in vitro and in vivo steps to generate sequence diversity by segment directed saturated mutagenesis and family shuffling. A high-throughput DNA chip-based procedure for the characterization and potentially the equalization of combinatorial libraries is also presented. Using these appro...
Article
Full-text available
Cytochrome P450 (CYP) enzymes represent a large superfamily that displays extraordinarily diverse substrate specificities. After a concise review about CYPs of the CYP1A subfamily, which plays a crucial role in procarcinogen activation, this paper presents segment-directed mutagenesis. This approach generates a library of random combinatorial mutan...
Article
Full-text available
Microsomal epoxide hydrolase (mEH) belongs to the superfamily of alpha/beta-hydrolase fold enzymes. A catalytic triad in the active centre of the enzyme hydrolyses the substrate molecules in a two-step reaction via the intermediate formation of an enzyme-substrate ester. Here we show that the mEH catalytic triad is composed of Asp226, Glu404 and Hi...
Article
An overview of current heterologous expression systems for xenobiotic metabolising enzymes is given with a special emphasis on the yeast expression system. In a first part, basic properties and relative advantages and drawbacks of each expression system are considered. The second part is dedicated to humanized yeast strains allowing human P450 expr...
Article
Full-text available
Two NADPH-cytochrome P450 reductase-encoding cDNAs were isolated from an Arabidopsis cDNA library by metabolic interference in a Saccharomyces cerevisiae mutant disrupted for its endogenous cpr1gene. ATR1 encodes a protein of 692 amino acids, whileATR2 encodes either a 712-residue protein (ATR2-1), or a 702-residue protein (ATR2-2) depending on the...
Article
The expression of functional heterologous P450s in yeast has shown that the yeast P450 reductase efficiently reduces helerologous P450s provided that a engineered strain overexpressing the P450 reductase be used. This led to the proposal that the P450 reductase of any species could reduce a P450 of any other species. In other words, there would not...
Article
An overview of current heterologous expression systems for xenobiotic metabolising enzymes is given with a special emphasis on the yeast expression system. In a first part, basic properties and relative advantages and drawbacks of each expression system are considered. The second part is dedicated to humanized yeast strains allowing human P450 expr...
Article
In many tissues from different species, pregnenolone and dehydroepiandrosterone (DHEA) are hydroxylated mainly at the 7 alpha position by a cytochrome P450 (P450)-containing microsomal enzyme complex. In addition, 7-hydroxysteroids have been shown to activate immune processes in mice. The reported production of 7 beta-hydroxypregnenolone and 7 beta...
Article
The metabolism of benzo[a]pyrene (B[a]P) and its proximate mutagen B[a]P-7,8-dihydrodiol (7,8-diol) was investigated in the presence of human microsomal epoxide hydrolase and P450 1A1, 1A2, 2C8, 2C9, 2C18, 2C19, 2D6 and 3A4 expressed in the yeast Saccharomyces cerevisiae. P450 1A1 had the highest turnover numbers for the formation of all B[a]P meta...
Article
The functional expression of human P450 3A4, NADPH-cytochromeP450-reductase (CPR) and cytochrome b5 (b5) in yeast allows to investigate the coupling mechanism in natural microsomal membranes instead of reconstitution in artificial micelles. Yeast chromosomal DNA was engineered by integration at the CPR1 locus (which encodes for the yeast CPR) an ex...
Article
In many tissues from different species, pregnenoIone and dehydroepiandrosterone (DHEA) are hydroxylated mainly at the 7α position by a cytochrome P450 (P450)-containing microsomal enzyme com-plex. In addition, 7-hydroxysteroids have been shown to activate immune processes in mice. The reported production of 7-hydroxypregnenolone and 7-hydroxy-DHE...
Article
Human cytochrome P450 1A1 (1A1) and microsomal epoxide hydrolase (mEH)-dependent metabolic activation of benzo[a]pyrene (BP) have been reconstituted with microsomes from yeast cells expressing the two enzymes. The formation of the postulated ultimate mutagen 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydro-BP, the so-called dio...
Article
The first generation of yeast expression systems relies on inducible expression cassettes borne by multicopy plasmids for production of unmodified human P450s and on the endogenous NADPH-P450 reductase to support activities. A second generation of engineered yeast involved targeted genomic modifications allowing overexpression of the yeast reductas...
Article
The first generation of yeast expression systems relies on inducible expression cassettes borne by multicopy plasmids for production of unmodified human P450s and on the endogenous NADPH-P450 reductase to support activities. A second generation of engineered yeast involved targeted genomic modifications allowing overexpression of the yeast reductas...
Article
Full-text available
Imidazoline binding sites (IBS) were proposed to be responsible for some of the pharmacological and therapeutic activities of imidazoline and related compounds and have been classified into two subtypes, I1BS and I2BS. Convergent studies attribute a role in central blood pressure regulation to the I1BS. In contrast, the function of I2BS remains unk...
Article
An expression library of hybrid cDNAs was constructed in vivo by homeologous recombination in yeast between human P450 1A1 and P450 1A2 sequences. Two clones exhibiting highly enhanced monooxygenase activities in vivo were selected. Chimera S12 includes the 88 N-terminal residues of P450 1A1 fused to the complementary part of the P450 1A2 sequence....
Article
Full-text available
Helianthus tuberosus cinnamate 4-hydroxylase (CYP73 or CA4H), a member of the P450 superfamily which catalyses the first oxidative step of the phenylpropanoid pathway in higher plants by transforming cinnamate into p-coumarate, was expressed in the yeast Saccharomyces cerevisiae. The PCR-amplified CA4H open reading frame was inserted into pYeDP60 u...
Article
Three natural allelic cDNAs coding for P-450 3A4, the major form in human liver, namely NF25, NF10 and hPCN1, have been expressed in Saccharomyces cerevisiae. NF25 and hPCN1 were functionally expressed in yeast microsomes, yielding proteins with an absorption maximum at 448 nm in the CO-reduced difference spectrum. Some catalytic activities and sub...
Chapter
Heterologous expression systems must ideally feature high, stable, reproducible and low-cost synthesis of any P450 under a native folding state, including full saturation by the hemin prosthetic group. Additionally, the host cell must be free of any interfering endogenous P450 activity and must offer a suitable P450 environment mimicking as closely...
Article
The usefulness of cDNA-directed expression of human hepatic P450s in yeast for the in vitro study of drug metabolism is emphasized. The major advantages of yeast expression are: (i) relatively high yields of heterologous P450 (approximately 5-10 nmol/l of culture medium) can be obtained; (ii) the expressed P450s are directly active in yeast microso...
Article
We have engineered yeast genomic DNA to construct a set of strains producing various relative amounts of yeast NADPH-P450 reductase (Yred) and human cytochrome b5 (Hb5). Expression of cDNAs encoding human P450 1A1, 1A2, 3A4, 19A and mouse P450 1A1 in the different oxido-reduction backgrounds thus constituted were achieved after strain transformatio...
Article
Human microsomal epoxide hydrolase and cytochrome P450 (P450) 1A1 were coexpressed in Saccharomyces cerevisiae from expression cassettes integrated respectively into the host chromosomal DNA and on a multicopy plasmid in a strain already overexpressing yeast NADPH-cytochrome P450 reductase (P450 reductase). A styrene-oxide-hydrolase activity (2 nmo...
Article
Human liver P450 NF25 (CYP3A4) had been previously expressed in Saccharomyces cerevisiae using the inducible GAL10-CYC1 promoter and the phosphoglycerate kinase gene terminator [Renaud, J. P., Cullin, C., Pompon, D., Beaune, P. and Mansuy, D. (1990) Eur. J. Biochem. 194, 889-896]. The use of an improved expression vector [Urban, P., Cullin, C. and...
Article
Introduction: Heterologous expression systems are tools increasingly used in simulating the P450-dependent metabolism of xenobiotic present in human liver. This led us to question the role that heterologous microsomal environment, and not only the P450 nature, can play in modulating reconstituted monooxygenase activities. Cytochromes P450 are not s...
Article
Human monoamine oxidases A and B were expressed under the control of a galactose inducible promoter in Saccharomyces cerevisiae. The two MAO isoenzymes were found located in the yeast mitochondrial outer membrane, probably in different orientations as suggested by controlled proteolysis experiments. A high level of both human MAO-A or -B activities...
Article
Cytochrome P-450s constitute a superfamily of mono-oxygenases which require the association with specific redox enzymes bound to the endoplasmic reticulum membrane for their activity. Conditions for the functional expression of these mammalian enzymes in yeast cells and the respective merits and limitations of currently used P-450 expression system...
Article
Hydroxyacid oxidase from rat kidney is an FMN-dependent enzyme that catalyzes the oxidation of L-alpha-hydroxy acids as well as, more slowly, that of L-alpha-amino acids. We report here a modified purification method for the enzyme, which is found to possess one cofactor per subunit of Mr 39,000. Determination of its N-terminal sequence suggests th...
Article
Fluoropyruvate inactivated oxidized flavocytochrome b2 (baker's yeast L-lactate dehydrogenase) in a biphasic process yielding convex semilog plots of residual activity versus time. At each reagent concentration, rate constants k1 and k2 for the two phases could be calculated by simulation studies using one of the schemes proposed by Ray and Koshlan...
Article
Full-text available
Bakers' yeast flavocytochrome b2 is a flavin-dependent L-2-hydroxy acid dehydrogenase which also exhibits transhydrogenase activity. When a reaction takes place between [2-3H]lactate and a halogenopyruvate, tritium is found in water and at the halogenolactate C2 position. When the halogenopyruvate undergoes halide ion elimination, tritium is also f...
Article
It has been shown that reduced flavocytochrome b2 not only catalyzes reduction of bromopyruvate [P. Urban, P.M. Alliel and F. Lederer (1983) Eur. J. Biochem. 134, 275-281] but also transforms it into pyruvate in a reductive elimination process. The dehydrohalogenation reaction also takes place when oxidized enzyme acts on bromolactate, but the reac...
Article
It is shown that, when baker's yeast flavocytochrome b2 is incubated with bromopyruvate in the presence of excess lactate, a transhydrogenation reaction takes place which produces bromolactate and pyruvate. The heme remains reduced during the reaction. It is further shown that reduced flavocytochrome b2 can catalyze the reduction of a number of oth...

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Projects (3)
Project
I am editing a special issue of Biomolecules (impact factor 4.9 in 2020) : Special Issue "New Insights into Cytochrome P450s". If you consider to present a manuscript for this special issue, please proceed to this webpage and ... just do it! https://www.mdpi.com/journal/biomolecules/special_issues/Cytochrome_P450s_Biomolecules You are most welcome to present your research or a review or an opinion paper on any aspect of cytochromes P450.
Project
The deep-learning artificial intelligence algorithm from DeepMind,, AlphaFold2, and its derivatives, AlphaFold-Multimer and AlphaFold-Advanced, are revolutionizing structural biology by the ease-to-get and very high accuracy of their 3D structure predictions. We are now using AlphaFold2 intensively to help us to get, at last, 3D structures of proteins and protein-protein complexes that, up to now, we could not get easily. Among other targets, we are using AlphaFold on P450 enzymes to help us to resolve discrepancies in biochemical data.
Project
Effect of ionic strength and chaotropic agents on the activity of NADPH-P450 reductase measured at two pHs: 7.4 and 6.7. Analysis of the molecular determinants of the redox reaction by using directed mutants of the human CPR