Microarray-based method for combinatorial library sequence mapping and characterization.

Here we describe a DNA-chip-based method for high-throughput sequence mapping. This involves competitive hybridization between short and differentially labeled fluorescent oligonucleotide probes and glass-supported PCR products. Competition between an excess of oligonucleotide probes targeting the same sequence segment improves sequence discrimination and reduces sensitivity to experimental conditions such as probe concentrations, hybridization, and washing temperatures and durations. The method was found to be particularly adapted to sequence mapping of combinatorial libraries obtained by DNA shuffling between members of a gene family. We present an application of this technique for the characterization of recombination biases in combinatorial libraries used in directed evolution.

Functional cloning, based on azole resistance in Saccharomyces cerevisiae, and characterization of Rhizopus nigricans redox carriers that are differentially involved in the P450-dependent response to progesterone stress.

The filamentous fungus Rhizopus nigricans responds to treatment with progesterone by inducing P450-associated redox carriers. Selection for azole resistance following expression of a cDNA library constructed with RNA from progesterone-treated R. nigricans in the yeast Saccharomyces cerevisiae led to the identification of CPR1-FL and CYB5-1 cDNAs, which code for functionally competent NADPH-cytochrome P450 reductase and cytochrome b5, respectively. The central region (CPR2-CS) of an additional reductase gene sharing 66% identity with CPR1-FL was cloned from progesterone-induced mRNA by RT-PCR, using primers based on consensus sequences. Northern analysis of the 2.1-kb transcripts revealed that, of the two cloned reductase genes, only CPR1-FL mRNA was strongly induced by progesterone; transcription of CYBS-1 and CPR2-CS mRNAs was not significantly affected. Analysis of the subcellular localization and function of the R. nigricans reductase in yeast indicated that the CPR1-FL cDNA and a derivative (CPR1-S) truncated at the first ATG codon gave rise to functionally equivalent products that were found in both cytosolic and microsomal fractions. In contrast, addition of an in-frame initiation codon at the 5' end of the CPR1-FL sequence resulted in localization of the activity mainly to the microsomes, and improved ketoconazole resistance but decreased NADPH-cytochrome c reductase activity in the host strain. These findings suggest that, of the three genes for P450-associated redox carriers investigated, only CPR1-FL is associated with the progesterone response and that its major transcript encodes a reductase that shows an unusual pattern of subcellular localization.

High efficiency family shuffling based on multi-step PCR and in vivo DNA recombination in yeast: statistical and functional analysis of a combinatorial library between human cytochrome P450 1A1 and 1A2.

The design of a family shuffling strategy (CLERY: Combinatorial Libraries Enhanced by Recombination in Yeast) associating PCR-based and in vivo recombination and expression in yeast is described. This strategy was tested using human cytochrome P450 CYP1A1 and CYP1A2 as templates, which share 74% nucleotide sequence identity. Construction of highly shuffled libraries of mosaic structures and reduction of parental gene contamination were two major goals. Library characterization involved multiprobe hybridization on DNA macro-arrays. The statistical analysis of randomly selected clones revealed a high proportion of chimeric genes (86%) and a homogeneous representation of the parental contribution among the sequences (55.8 +/- 2.5% for parental sequence 1A2). A microtiter plate screening system was designed to achieve colorimetric detection of polycyclic hydrocarbon hydroxylation by transformed yeast cells. Full sequences of five randomly picked and five functionally selected clones were analyzed. Results confirmed the shuffling efficiency and allowed calculation of the average length of sequence exchange and mutation rates. The efficient and statistically representative generation of mosaic structures by this type of family shuffling in a yeast expression system constitutes a novel and promising tool for structure-function studies and tuning enzymatic activities of multicomponent eucaryote complexes involving non-soluble enzymes.

P450BM-3: absolute configuration of the primary metabolites of palmitic acid.

P450BM-3, a catalytically self-sufficient, soluble bacterial P450, contains on the same polypeptide a heme domain and a reductase domain. P450BM-3 catalyzes the oxidation of short- and long-chain, saturated and unsaturated fatty acids. The three-dimensional structure of the heme domain both in the absence and in the presence of fatty acid substrates has been determined; however, the fatty acid in the substrate-bound form is not adequately close to the heme iron to permit a prediction regarding the stereoselectivity of oxidation. In the case of long-chain fatty acids, the products can also serve as substrate and be metabolized several times. In the current study, we have determined the absolute configuration of the three primary products of palmitic acid hydroxylation (15-, 14-, and 13-OH palmitic acid). While the 15- and 14-hydroxy compounds are produced in a highly stereoselective manner (98% R, 2% S), the 13-hydroxy is a mixture of 72% R and 28% S. We have also examined the binding of these three hydroxy acids to P450BM-3 and shown that only two of them (14-OH and 13-OH palmitic acid) can bind to and be further metabolized by P450BM-3. The results indicate that in contrast to the flexibility of palmitoleic acid bound to the oxidized enzyme, palmitic acid is rigidly bound in the active site during catalytic turnover.

Thr268 in substrate binding and catalysis in P450BM-3.

Members of the gene superfamily of proteins called "P450" catalyze monooxygenation reactions that require an input of two electrons and a molecule of oxygen per catalytic cycle. These proteins are widely distributed among living organisms, from bacteria to human. P450BM-3, a soluble protein isolated from Bacillus megaterium, is self-sufficient, containing P450 and reductase domains on the same polypeptide. P450BM-3 catalyzes the hydroxylation of various fatty acids at omega-1, omega-2, and omega-3 positions, as well as epoxidations of double bonds. We have constructed the active-site mutant, T268A, and analyzed the effect on arachidonic acid and palmitic acid oxidation. Data indicate that the mutation changes the coupling (ratio of NADPH consumed versus product formed) for both arachidonic acid and palmitic acid oxidation. We have also analyzed cumene hydroperoxide-driven reactions and shown that they are unaffected by this mutation. These data, as well as fatty acid binding studies, support the hypothesis of a role of the I-helix residue, T268, in maintaining fatty acid substrates in the correct position for productive hydroxylation during the catalytic cycle of this enzyme.

Reconstitution of the fatty acid hydroxylase activity of cytochrome P450BM-3 utilizing its functional domains.

Cytochrome P450BM-3, a catalytically self-sufficient fatty acid monooxygenase from Bacillus megaterium, is a multidomain protein containing heme, FAD, and FMN. Previous attempts to reconstitute the fatty acid monooxygenase activity of intact P450BM-3 utilizing equimolar concentrations of the separate heme (BMP) and reductase (BMR) domains, have been unsuccessful because two-electron reduced FMN, which rapidly accumulates, is incapable of electron transfer to the heme iron. The present study of the reconstitution of the monooxygenase activity of P450BM-3 utilized combinations of the different functional domains of P450BM-3. For this purpose, the FAD/NADPH- and FMN-binding domains of P450BM-3 as well as the combination of the heme- and FMN-binding domains (BMP/FMN) have been expressed and purified. The reconstitution systems, consisting of either BMP/FMN and FAD domains or BMP, FMN, and FAD domains, were still less effective than the holoenzyme, P450BM-3, but were much more effective than a system consisting of BMP and BMR. The maximal rate of oxidation of palmitic acid by the newly developed reconstitution systems is still only approximately 5% of the activity of the holoenzyme. The reconstitution systems produced omega-1, omega-2, and omega-3 monohydroxy palmitic acid, but not the secondary products of palmitic acid hydroxylation observed with the holoenzyme. The physical cause of the inability to reconstitute fully the maximal activity of the holoenzyme as well as the lack of secondary product formation is not presently understood.

Simulation of human xenobiotic metabolism in microorganisms. Yeast a good compromise between E. coli and human cells.

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 expression in a tailored redox environment and to the possibility to use such strains to simulate complex metabolisms involving a combination of phase I and phase II reactions. The last part presents how the association of numeric simulation to yeast expression can help in understanding rules controlling metabolic profiles in xenobiotic-acting multienzymatic systems.

P450BM-3; a tale of two domains--or is it three?

Over 400 P450s have been identified to date in prokaryotes and eukaryotes, plants and animals, mitochondria and endoplasmic reticulum. These enzymes function in areas such as metabolism and steroidogenesis. The eukaryotic members of this gene superfamily of proteins have proved difficult to study because of the hydrophobic nature of their substrates, their various redox partners, and membrane association. To better understand the structure/function relationship of P450s-what determines substrate specificity and selectivity, what determines redox-partner binding, and which regions are involved in membrane binding-we have compared the three crystallized, soluble bacterial P450s (two class I and one class II) and a model of a steroidogenic, eukaryotic P450 (P450arom), to define which structural elements form a conserved structural fold for P450s, what determines specificity of substrate binding and redox-partner binding, and which regions are potentially involved in membrane association. We believe that there is a conserved structural fold for all P450s that can be used to model those P450s that prove intransigent to structural determination. However, although there appears to be a conserved structural core among P450s, there is sufficient sequence variability that no two P450s are structurally identical. NADPH-P450 reductase transfers electrons from NADPH to P450 during the P450 catalytic cycle. This enzyme has usually been thought of as a simple globular protein; however, sequence analysis has shown that NADPH-P450 reductase is related to two separate flavoprotein families, ferredoxin nucleotide reductase (FNR) and flavodoxin. Recent studies by Wolff and his colleagues have shown that the FAD-binding FNR domain and FMN-binding flavodoxin domain of human NADPH-P450 reductase can be independently expressed in Escherichia coli. The subdomains can be used to reconstitute, however poorly, the monooxygenase activity of the P450 system. We have been utilizing the reductase domain of P450BM-3 to study the mechanism of electron transfer from NADPH to P450 in this complex multidomain protein. We have overexpressed both the FNR subdomain and the flavodoxin subdomain in E. coli and fully reconstituted the cytochrome c reductase activity of this enzyme. Our studies have shown that electron transfer from NADPH through the reductase domain to the P450 requires shuttling of the FMN subdomain between the reductase subdomain and the P450. Studies of the factors that control the molecular recognition and interaction among these three proteins are complicated by the weakness of the association and changes in the strength of the interaction depending on the redox state of each of the components. How these structural and mechanistic studies of a soluble bacterial P450 can be extended to gain a better understanding of the control of membrane-bound eukaryotic P450-dependent redox systems is discussed.

An active site substitution, F87V, converts cytochrome P450 BM-3 into a regio- and stereoselective (14S,15R)-arachidonic acid epoxygenase.

Cytochrome P450 BM-3 catalyzes the high turnover regio- and stereoselective metabolism of arachidonic and eicosapentaenoic acids. To map structural determinants of productive active site fatty acid binding, we mutated two amino acid residues, arginine 47 and phenylalanine 87, which flank the surface and heme ends of the enzyme's substrate access channel, respectively. Replacement of arginine 47 with glutamic acid resulted in a catalytically inactive mutant. Replacement of arginine 47 with alanine yielded a protein with reduced substrate binding affinity and arachidonate sp3 carbon hydroxylation activity (72% of control wild type). On the other hand, arachidonic and eicosapentaenoic acid epoxidation was significantly enhanced (154 and 137%, of control wild type, respectively). As with wild type, the alanine 47 mutant generated (18R)-hydroxyeicosatetraenoic, (14S,15R)-epoxyeicosatrienoic, and (17S,18R)-epoxyeicosatetraenoic acids nearly enantiomerically pure. Replacement of phenylalanine 87 with valine converted cytochrome P450 BM-3 into a regio- and stereoselective arachidonic acid epoxygenase ((14S,15R)-epoxyeicosatrienoic acid, 99% of total products). Conversely, metabolism of eicosapentaenoic acid by the valine 87 mutant yielded a mixture of (14S,15R)- and (17S,18R)-epoxyeicosatetraenoic acids (26 and 69% of total, 94 and 96% optical purity, respectively). Finally, replacement of phenylalanine 87 with tyrosine yielded an inactive protein. We propose that: (a) fatty acid oxidation by P450 BM-3 is incompatible with the presence of residues with negatively charged side chains at the surface opening of the substrate access channel or a polar aromatic side chain in the vicinity of the heme iron; (b) the high turnover regio- and stereoselective metabolism of arachidonic and eicosapentaenoic acids involves charge-dependent anchoring of the fatty acids at the mouth of the access channel by arginine 47, as well as steric gating of the heme-bound oxidant by phenylalanine 87; and (c) substrate binding coordinates, as opposed to oxygen chemistries, are the determining factors responsible for reaction rates, product chemistry, and, thus, catalytic outcome.

The highly stereoselective oxidation of polyunsaturated fatty acids by cytochrome P450BM-3.

Cytochrome P450BM-3 catalyzes NADPH-dependent metabolism of arachidonic acid to nearly enantiomerically pure 18(R)-hydroxyeicosatetraenoic acid and 14(S), 15(R)-epoxyeicosatrienoic acid (80 and 20% of total products, respectively). P450BM-3 oxidizes arachidonic acid with a rate of 3.2 +/- 0.4 micromol/min/nmol at 30 degrees C, the fastest ever reported for an NADPH-dependent, P450-catalyzed reaction. Fatty acid, oxygen, and NADPH are utilized in an approximately 1:1:1 molar ratio, demonstrating efficient coupling of electron transport to monooxygenation. Eicosapentaenoic and eicosatrienoic acids, two arachidonic acid analogs that differ in the properties of the C-15-C-18 carbons, are also actively metabolized by P450BM-3 (1.4 +/- 0.2 and 2.9 +/- 0.1 micromol/min/nmol at 30 degrees C, respectively). While the 17,18-olefinic bond of eicosapentaenoic acid is epoxidized with nearly absolute regio- and stereochemical selectivity to 17(S),18(R)-epoxyeicosatetraenoic acid (>/=99% of total products, 97% optical purity), P450BM-3 is only moderately regioselective during hydroxylation of the eicosatrienoic acid omega-1, omega-2, and omega-3 sp3 carbons, with 17-, 18-, and 19-hydroxyeicosatrienoic acid formed in a ratio of 2.4:2.2:1, respectively. Based on the above and on a model of arachidonic acid-bound P450BM-3, we propose: 1) the formation by P450BM-3 of a single oxidant species capable of olefinic bond epoxidation and sp3 carbon hydroxylation and 2) that product chemistry and, thus, catalytic outcome are critically dependent on active site spatial coordinates responsible for substrate binding and productive orientation between heme-bound active oxygen and acceptor carbon bond(s).

The flavoprotein domain of P450BM-3: expression, purification, and properties of the flavin adenine dinucleotide- and flavin mononucleotide-binding subdomains.

P450BM-3 is a self-sufficient fatty acid monooxygenase that can be expressed in Escherichia coli as either the holoenzyme or as the individual hemo- and flavoprotein domains. The flavoprotein domain (BMR) of P450BM-3 is soluble and contains an equimolar ratio of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) and is functionally analogous to microsomal nicotinamide adenine dinucleotide phosphate (NADPH)-P450 reductases. These reductases have been proposed to have evolved through a fusion of genes encoding simple flavin-containing electron-transport proteins [Porter, T. D. (1991) Trends Biochem. Sci. 16, 154-158]. The gene encoding BMR has been divided into the coding regions for the FAD/NADPH- and FMN-binding domains. These proteins were overexpressed in E. coli and both domains were found to contain not less than 0.9 +/- 0.05 mol of FAD or FMN/mol of protein. Compared to BMR, the electron-accepting properties of the recombinant flavin domains were mainly conserved. Titration of the FMN domain with sodium dithionite resulted in the conversion of the protein to the fully reduced FMNH2 form without accumulation of intermediate semiquinone forms; however, a similar titration of the FAD domain gave clear evidence for the presence of a neutral, blue flavin semiquinone during the reduction. Titrations of the reduced forms of the domains with artificial electron acceptors indicated that the electron-transferring properties of both the FAD- and FMN domains were also conserved. The rate constants of reoxidation of the fully reduced FAD and FMN domains by molecular oxygen at 20 degrees C were found to be 2.5 and 0.1 min-1, respectively. The cytochrome c reductase activity of BMR could be fully reconstituted with the individual domains. The data presented support the hypothesis that BMR has a discrete multidomain structure.

Cloning and characterization of a yeast cytochrome b5-encoding gene which suppresses ketoconazole hypersensitivity in a NADPH-P-450 reductase-deficient strain.

Cytochrome P-450 (Cyp) 51 or lanosterol-C14-demethylase is the main target for antifungal compounds of the triazole family like ketoconazole (Kz). Disruption of the associated NADPH-P-450 reductase-encoding gene (YRED) is not lethal, but decreases by about 20-fold the Kz resistance (KzR) of wild-type (wt) Saccharomyces cerevisiae. Transformation of a YRED-disrupted strain by a yeast genomic library based on a multicopy vector allowed us to identify a suppressor of Kz hypersensitivity. Deletion analysis of the 5-kb cloned fragment indicated that yeast cytochrome b5-encoding gene (CYB5), which encodes a 120-amino-acid (aa) protein, is required and sufficient for the suppressor effect. The encoded polypeptide shares about 30% aa identity with mammalian cytochromes b5 (Cyb5). CYB5 disruption and tetrad analysis demonstrate that yeast Cyb5 is not required for growth in a Yred+ strain. Determination of the microsomal content of b-type cytochromes by differential spectra indicated the presence of a strongly decreased or null Cyb5 level in the disrupted strain. This confirms that we have cloned the gene encoding the major microsomal form of Cyb5 which appears not to be essential. Minor Cyb5 isoforms could also be present in yeast or other redox proteins could substitute for the pleiotropic roles of Cyb5 in the sterol and lipid biosynthesis pathways.

Recombinant yeast in drug metabolism.

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 microsomes, allowing the determination of specific catalytic activities of individual isoforms, which is a prerequisite for the prediction of metabolic pathways for new drug candidates; (iii) transformed yeast microsomes can also be used to study the specific affinity of individual P450s for various substrates and the formation of P450-metabolite complexes by difference visible spectroscopy; such studies can help to predict drug interactions. The advantages of expression in yeast with respect to biochemical studies of drug metabolism are illustrated with data about P450 NF25 (P450 3A4), the major form of human liver. Expressed P450 NF25 is obtained in a functionally active state, and some specific catalytic activities observed in liver microsomes could be reproduced directly with transformed yeast microsomes. The use of genomically modified yeast strains coexpressing human cytochrome b5 and/or overexpressing yeast P450-reductase allowed us to optimize these catalytic activities. In particular, this coexpression system was useful in the study of the in vitro formation of a P450 NF25 Fe(II)-RNO complex. Such inhibitory complexes have been implied in numerous drug interactions involving P450 3A4.

Enhanced in vivo monooxygenase activities of mammalian P450s in engineered yeast cells producing high levels of NADPH-P450 reductase and human cytochrome b5.

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 transformation by plasmid-based P450-encoding expression cassettes. The results indicate that the level of Yred strongly affects all activities tested. In contrast, the amount of Hb5 affects activities in a manner that is dependent both on the P450 isoform considered and the Yred level. In a strain containing optimized amounts of Hb5 and Yred, human P450 3A4-specific testosterone-6 beta-hydroxylase activity can be enhanced as much as 73-fold in comparison with the activity observed in a wild-type strain. Bioconversion of sterols or xenobiotics was easily achieved in vivo using this new co-expression system.

Optimization of yeast-expressed human liver cytochrome P450 3A4 catalytic activities by coexpressing NADPH-cytochrome P450 reductase and cytochrome b5.

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 Pompon, D. (1990) Biochimie 72, 463-472] increased the amounts of P450 NF25 produced/culture medium by a factor of five, yielding up to 10 nmol/l. The availability of recently developed host cells that simultaneously overexpress yeast NADPH-P450 reductase and/or express human liver cytochrome b5, obtained through stable integration of the corresponding coding sequences into the yeast genome, led to biotechnological systems with much higher activities of yeast-expressed P450 NF25 and with much better ability to form P450 NF25-iron-metabolite complexes. 9-fold, 8-fold, and 30-fold rate increases were found respectively for nifedipine 1,4-oxidation, lidocaine N-deethylation and testosterone 6 beta-hydroxylation between P450 NF25-containing yeast microsomes from the basic strain and from the strain that both overexpresses yeast NADPH-P450 reductase and expresses human cytochrome b5. Even higher turnovers (15-fold, 20-fold and 50-fold rate increases) were obtained using P450 NF25-containing microsomes from the yeast just overexpressing yeast NADPH-P450 reductase in the presence of externally added, purified rabbit liver cytochrome b5. This is explained by the fact that the latter strain contained the highest level of NADPH-P450 reductase activity. It is noteworthy that for the three tested substrates, the presence of human or rabbit cytochrome b5 always showed a stimulating effect on the catalytic activities and this effect was saturable. Indeed, addition of rabbit cytochrome b5 to microsomes from a strain expressing human cytochrome b5 did not further enhance the catalytic rates. The yeast expression system was also used to study the formation of a P450-NF25-iron-metabolite complex. A P450 Fe(II)-(RNO) complex was obtained upon oxidation of N-hydroxyamphetamine, catalyzed by P450-NF25-containing yeast microsomes. In microsomes from the basic strain expressing P450 NF25, 10% of the starting P450 NF25 was transformed into this metabolite complex, whereas more than 80% of the starting P450 NF25 led to complex formation in microsomes from the strain overexpressing yeast NADPH-P450 reductase.(ABSTRACT TRUNCATED AT 250 WORDS)