Bacterial Cytochrome P450 Monooxygenases Research Articles

The oxidation of steroid derivatives by the CYP125A6 and CYP125A7 enzymes from Mycobacterium marinum

The members of the bacterial cytochrome P450 (CYP) monooxygenase family CYP125, catalyze the oxidation of steroid derivatives including cholesterol and phytosterols, as the initial activating step in their catabolism. However, several bacterial species contain multiple genes encoding CYP125 enzymes and other CYP enzymes which catalyze cholesterol/cholest-4-en-3-one hydroxylation. An important question is why these bacterium have more than one enzyme with overlapping substrate ranges capable of catalyzing the terminal oxidation of the alkyl chain of these sterols. To further understand the role of these enzymes we investigated CYP125A6 and CYP125A7 from Mycobacterium marinum with various cholesterol analogues. These have modifications on the A and B rings of the steroid and we assessed the substrate binding and catalytic activity of these with each enzyme. CYP125A7 gave similar results to those reported for the CYP125A1 enzyme from M. tuberculosis. Differences in the substrate binding and catalytic activity with the cholesterol analogues were observed with CYP125A6. For example, while cholesteryl sulfate could bind to both enzymes it was only oxidized by CYP125A6 and not by CYP125A7. CYP125A6 generated higher levels of metabolites with the majority of C-3 and C-7 substituted cholesterol analogues such 7-ketocholesterol. However, 5α-cholestan-3β-ol was only oxidized by CYP125A7 enzyme. The cholest-4-en-3-one and 7-ketocholesterol-bound forms of the CYP125A6 and CYP125A7 enzymes were modelled using AlphaFold. The structural models highlighted differences in the binding modes of the steroid derivatives within the same enzyme. Significant changes in the binding mode of the steroids between these CYP125 enzymes and other bacterial cholesterol oxidizing enzymes, CYP142A3 and CYP124A1, were also seen. Despite this, all these models predicted the selectivity for terminal methyl hydroxylation, in agreement with the experimental data.

Enhanced metabolism of 2,3′,4,4′,5-pentachlorobiphenyl (CB118) by bacterial cytochrome P450 monooxygenase mutants of Bacillus megaterium

Bacterial cytochrome P450 monooxygenase P450BM3 is a promising enzyme to provide novel substrate specificity and enhanced enzymatic activity. The wild type (WT) has been shown to metabolize the widely distributed polychlorinated biphenyl (PCB) 2,3′,4,4′,5-pentachlorobiphenyl (CB118) to hydroxylated metabolites. However, this reaction requires the coexistence of perfluoroalkyl carboxylic acids (PFCAs). To locate P450BM3 mutants metabolizing CB118 without PFCAs, mutations were selected from amino acids comprising the substrate-binding cavity and the substrate entrance. The mutant A264G showed enhanced hydroxylation activities compared to the WT for the production of five hydroxylated metabolites. Perfluorooctanoic acid addition provided the highest activity, as found in the WT. The docking model of A264G and CB118 indicated that the enlargement of the space above the heme brought CB118 close to the heme, resulting in high activity. In contrast, the mutants L188Q, QG, LVQ, and GVQ, which contain the L188Q mutation, showed higher activity than WT even without PFCAs. Docking models revealed that the closed form found in substrate binding was induced by the L188Q mutation in the substrate non-binding state of the mutants. These mutants are promising for bioremediation of PCBs using enhanced metabolizing activities.

The oxidation of cholesterol derivatives by the CYP124 and CYP142 enzymes from Mycobacterium marinum

The CYP124 and CYP142 families of bacterial cytochrome P450 monooxygenases (CYPs), catalyze the oxidation of methyl branched lipids, including cholesterol, as one of the initial activating steps in their catabolism. Both enzymes are reported to supplement the CYP125 family of P450 enzymes. These CYP125 enzymes are found in the same bacteria, and are the primary cholesterol/cholest-4-en-3-one metabolizing enzymes. To further understand the role of the CYP124 and CYP142 cytochrome P450s we investigated the Mycobacterium marinum enzymes, MmarCYP124A1 and CYP142A3, with various cholesterol analogs with modifications on the A and B rings of the steroid. We assessed the substrate binding and catalytic activity of each enzyme. Neither enzyme could bind or oxidize cholesteryl acetate or 3,5-cholestadiene, which have modifications at the C3 hydroxyl moiety of cholesterol. The CYP142 enzyme was better able to accommodate and oxidize cholesterol analogs which have changes on the A/B rings including cholesterol-5α,6α-epoxide and diastereomers of 5-cholestan-3-ol. The CYP124 enzyme was more tolerant of changes at C7 of the cholesterol B ring, e.g., 7-ketocholesterol than in the A ring. The selectivity for oxidation at the ω-carbon of a branched chain was observed in all steroids that were oxidized. The 7-ketocholesterol-bound MmarCYP124A1 enzyme from M. marinum, was structurally characterized by X-ray crystallography to 1.81 Å resolution. The 7-ketocholesterol-bound X-ray crystal structure of the MmarCYP124A1 enzyme revealed that the substrate binding mode of this cholesterol derivative was altered compared to those observed with other non-steroidal ligands. The structure provided an explanation for the selectivity of the enzyme for terminal methyl hydroxylation.

Characterization of a Virally Encoded Flavodoxin That Can Drive Bacterial Cytochrome P450 Monooxygenase Activity.

Flavodoxins are small electron transport proteins that are involved in a myriad of photosynthetic and non-photosynthetic metabolic pathways in Bacteria (including cyanobacteria), Archaea and some algae. The sequenced genome of 0305φ8-36, a large bacteriophage that infects the soil bacterium Bacillus thuringiensis, was predicted to encode a putative flavodoxin redox protein. Here we confirm that 0305φ8-36 phage encodes a FMN-containing flavodoxin polypeptide and we report the expression, purification and enzymatic characterization of the recombinant protein. Purified 0305φ8-36 flavodoxin has near-identical spectral properties to control, purified Escherichia coli flavodoxin. Using in vitro assays we show that 0305φ8-36 flavodoxin can be reconstituted with E. coli flavodoxin reductase and support regio- and stereospecific cytochrome P450 CYP170A1 allyl-oxidation of epi-isozizaene to the sesquiterpene antibiotic product albaflavenone, found in the soil bacterium Streptomyces coelicolor. In vivo, 0305φ8-36 flavodoxin is predicted to mediate the 2-electron reduction of the β subunit of phage-encoded ribonucleotide reductase to catalyse the conversion of ribonucleotides to deoxyribonucleotides during viral replication. Our results demonstrate that this phage flavodoxin has the potential to manipulate and drive bacterial P450 cellular metabolism, which may affect both the host biological fitness and the communal microbiome. Such a scenario may also be applicable in other viral-host symbiotic/parasitic relationships.

Identification of Fungal Limonene-3-Hydroxylase for Biotechnological Menthol Production.

More than 30,000 tons of menthol are produced every year as a flavor and fragrance compound or as a medical component. So far, only extraction from plant material and chemical synthesis are possible. An alternative approach for menthol production could be a biotechnological-chemical process with ideally only two conversion steps, starting from (+)-limonene, which is a side product of the citrus processing industry. The first step requires a limonene-3-hydroxylase (L3H) activity that specifically catalyzes hydroxylation of limonene at carbon atom 3. Several protein engineering strategies have already attempted to create limonene-3-hydroxylases from bacterial cytochrome P450 monooxygenases (CYPs, or P450s), which can be efficiently expressed in bacterial hosts. However, their regiospecificity is rather low compared to that of the highly selective L3H enzymes from the biosynthetic pathway for menthol in Mentha species. The only naturally occurring limonene-3-hydroxylase activity identified in microorganisms so far was reported for a strain of the black yeast-like fungus Hormonema sp. in South Africa. We have discovered additional fungi that can catalyze the intended reaction and identified potential CYP-encoding genes within the genome sequence of one of the strains. Using heterologous gene expression and biotransformation experiments in yeasts, we were able to identify limonene-3-hydroxylases from Aureobasidium pullulans and Hormonema carpetanum Further characterization of the A. pullulans enzyme demonstrated its high stereospecificity and regioselectivity, its potential for limonene-based menthol production, and its additional ability to convert α- and β-pinene to verbenol and pinocarveol, respectively.IMPORTANCE (-)-Menthol is an important flavor and fragrance compound and furthermore has medicinal uses. To realize a two-step synthesis starting from renewable (+)-limonene, a regioselective limonene-3-hydroxylase enzyme is necessary. We identified enzymes from two different fungi which catalyze this hydroxylation reaction and represent an important module for the development of a biotechnological process for (-)-menthol production from renewable (+)-limonene.

Riboflavin Is Directly Involved in N-Dealkylation Catalyzed by Bacterial Cytochrome P450 Monooxygenases.

Like a vast number of enzymes in nature, bacterial cytochrome P450 monooxygenases require an activated form of flavin as a cofactor for catalytic activity. Riboflavin is the precursor of FAD and FMN that serves as indispensable cofactor for flavoenzymes. In contrast to previous notions, herein we describe the identification of an electron-transfer process that is directly mediated by riboflavin for N-dealkylation by bacterial P450 monooxygenases. The electron relay from NADPH to riboflavin and then via activated oxygen to heme was proposed based on a combination of X-ray crystallography, molecular modeling and molecular dynamics simulation, site-directed mutagenesis and biochemical analysis of representative bacterial P450 monooxygenases. This study provides new insights into the electron transfer mechanism in bacterial P450 enzyme catalysis and likely in yeasts, fungi, plants and mammals.

Heterologous caffeic acid biosynthesis in Escherichia coli is affected by choice of tyrosine ammonia lyase and redox partners for bacterial Cytochrome P450

BackgroundCaffeic acid is industrially recognized for its antioxidant activity and therefore its potential to be used as an anti-inflammatory, anticancer, antiviral, antidiabetic and antidepressive agent. It is traditionally isolated from lignified plant material under energy-intensive and harsh chemical extraction conditions. However, over the last decade bottom-up biosynthesis approaches in microbial cell factories have been established, that have the potential to allow for a more tailored and sustainable production. One of these approaches has been implemented in Escherichia coli and only requires a two-step conversion of supplemented l-tyrosine by the actions of a tyrosine ammonia lyase and a bacterial Cytochrome P450 monooxygenase. Although the feeding of intermediates demonstrated the great potential of this combination of heterologous enzymes compared to others, no de novo synthesis of caffeic acid from glucose has been achieved utilizing the bacterial Cytochrome P450 thus far.ResultsThe herein described work aimed at improving the efficiency of this two-step conversion in order to establish de novo caffeic acid formation from glucose. We implemented alternative tyrosine ammonia lyases that were reported to display superior substrate binding affinity and selectivity, and increased the efficiency of the Cytochrome P450 by altering the electron-donating redox system. With this strategy we were able to achieve final titers of more than 300 µM or 47 mg/L caffeic acid over 96 h in an otherwise wild type E. coli MG1655(DE3) strain with glucose as the only carbon source. We observed that the choice and gene dose of the redox system strongly influenced the Cytochrome P450 catalysis. In addition, we were successful in applying a tethering strategy that rendered even a virtually unproductive Cytochrome P450/redox system combination productive.ConclusionsThe caffeic acid titer achieved in this study is about 10% higher than titers reported for other heterologous caffeic acid pathways in wildtype E. coli without l-tyrosine supplementation. The tethering strategy applied to the Cytochrome P450 appears to be particularly useful for non-natural Cytochrome P450/redox partner combinations and could be useful for other recombinant pathways utilizing bacterial Cytochromes P450.

Biomimetic and Biocatalytic Synthesis of Bruceol

AbstractThe first total synthesis of bruceol has been achieved using a biomimetic cascade cyclization initiated by a stereoselective Jacobsen–Katsuki epoxidation (and kinetic resolution) of racemic protobruceol‐I. A bacterial cytochrome P450 monooxygenase was also found to catalyze the conversion of protobruceol‐I into bruceol. The first full analysis of the NMR data of natural bruceol suggested that “isobruceol” was a previously unrecognized natural product also isolated from Philotheca brucei. This was confirmed by the re‐isolation, synthesis, and X‐ray analysis of isobruceol. In total, eight stereoisomers and structural isomers of bruceol have been synthesized in a highly divergent approach.

Biomimetic and Biocatalytic Synthesis of Bruceol.

The first total synthesis of bruceol has been achieved using a biomimetic cascade cyclization initiated by a stereoselective Jacobsen-Katsuki epoxidation (and kinetic resolution) of racemic protobruceol-I. A bacterial cytochrome P450 monooxygenase was also found to catalyze the conversion of protobruceol-I into bruceol. The first full analysis of the NMR data of natural bruceol suggested that "isobruceol" was a previously unrecognized natural product also isolated from Philotheca brucei. This was confirmed by the re-isolation, synthesis, and X-ray analysis of isobruceol. In total, eight stereoisomers and structural isomers of bruceol have been synthesized in a highly divergent approach.

A Stable Artificial Multienzymatic Complex Using a Heterotrimeric Protein From Metallosphaera sedula.

Bacterial cytochrome P450 monooxygenases (P450s) are promising biocatalysts for chemical syntheses because they catalyze a variety of oxidations on non-activated hydrocarbons using O2 . However, the requirement of two auxiliary proteins, an electron transfer protein and a reductase, for the catalysis is a major bottleneck for in vitro applications of these monooxygenases. The authors previous study showed that artificial assembly of a bacterial P450 with its auxiliary proteins using a heterotrimeric proliferating cell nuclear antigen (PCNA) from Sulfolobus solfataricus yields a self-sufficient P450, but partial dissociation of P450 from the complex at catalytic concentrations reduces the apparent specific activity of this self-sufficient P450. In this study, a Metallosphaera sedula PCNA is used, which is currently the most stable heterotrimeric PCNA, to assemble a bacterial P450 with its auxiliary proteins at submicromolar protein concentrations. The apparent specific monooxygenase activity of the M. sedula PCNA-assembled P450 with auxiliary proteins is saturated at protein concentrations of 40 nM, and is 2.1-fold higher than that of the S. solfataricus PCNA-assembled P450. Therefore, M. sedula PCNA represents a versatile tool to facilitate multiple enzymatic reactions, including the P450 monooxygenase system.

Identification and characterization of a bacterial cytochrome P450 monooxygenase catalyzing the 3-nitration of tyrosine in rufomycin biosynthesis

Rufomycin is a circular heptapeptide with anti-mycobacterial activity and is produced by Streptomyces atratus ATCC 14046. Its structure contains three non-proteinogenic amino acids, N-dimethylallyltryptophan, trans-2-crotylglycine, and 3-nitrotyrosine (3NTyr). Although the rufomycin structure was already reported in the 1960s, its biosynthesis, including 3NTyr generation, remains unclear. To elucidate the rufomycin biosynthetic pathway, we assembled a draft genome sequence of S. atratus and identified the rufomycin biosynthetic gene cluster (ruf cluster), consisting of 20 ORFs (rufA-rufT). We found a putative heptamodular nonribosomal peptide synthetase encoded by rufT, a putative tryptophan N-dimethylallyltransferase encoded by rufP, and a putative trimodular type I polyketide synthase encoded by rufEF Moreover, the ruf cluster contains an apparent operon harboring putative cytochrome P450 (rufO) and nitric oxide synthase (rufN) genes. A similar operon, txtDE, is responsible for the formation of 4-nitrotryptophan in thaxtomin biosynthesis; the cytochrome P450 TxtE catalyzes the 4-nitration of Trp. Therefore, we hypothesized that RufO should catalyze the Tyr 3-nitration. Disruption of rufO abolished rufomycin production by S. atratus, which was restored when 3NTyr was added to the culture medium of the disruptant. Recombinant RufO protein exhibited Tyr 3-nitration activity both in vitro and in vivo Spectroscopic analysis further revealed that RufO recognizes Tyr as the substrate with a dissociation constant of ∼0.1 μm These results indicate that RufO is an unprecedented cytochrome P450 that catalyzes Tyr nitration. Taken together with the results of an in silico analysis of the ruf cluster, we propose a rufomycin biosynthetic pathway in S. atratus.

Engineering of versatile redox partner fusions that support monooxygenase activity of functionally diverse cytochrome P450s

Most bacterial cytochrome P450 monooxygenases (P450s or CYPs) require two redox partner proteins for activity. To reduce complexity of the redox chain, the Bacillus subtilis flavodoxin YkuN (Y) was fused to the Escherichia coli flavodoxin reductase Fpr (R), and activity was tuned by placing flexible (GGGGS)n or rigid ([E/L]PPPP)n linkers (n = 1–5) in between. P-linker constructs typically outperformed their G-linker counterparts, with superior performance of YR-P5, which carries linker ([E/L]PPPP)5. Molecular dynamics simulations demonstrated that ([E/L]PPPP)n linkers are intrinsically rigid, whereas (GGGGS)n linkers are highly flexible and biochemical experiments suggest a higher degree of separation between the fusion partners in case of long rigid P-linkers. The catalytic properties of the individual redox partners were best preserved in the YR-P5 construct. In comparison to the separate redox partners, YR-P5 exhibited attenuated rates of NADPH oxidation and heme iron (III) reduction, while coupling efficiency was improved (28% vs. 49% coupling with B. subtilis CYP109B1, and 44% vs. 50% with Thermobifida fusca CYP154E1). In addition, YR-P5 supported monooxygenase activity of the CYP106A2 from Bacillus megaterium and bovine CYP21A2. The versatile YR-P5 may serve as a non-physiological electron transfer system for exploitation of the catalytic potential of other P450s.

Prospecting Biotechnologically-Relevant Monooxygenases from Cold Sediment Metagenomes: An In Silico Approach.

The goal of this work was to identify sequences encoding monooxygenase biocatalysts with novel features by in silico mining an assembled metagenomic dataset of polar and subpolar marine sediments. The targeted enzyme sequences were Baeyer–Villiger and bacterial cytochrome P450 monooxygenases (CYP153). These enzymes have wide-ranging applications, from the synthesis of steroids, antibiotics, mycotoxins and pheromones to the synthesis of monomers for polymerization and anticancer precursors, due to their extraordinary enantio-, regio-, and chemo- selectivity that are valuable features for organic synthesis. Phylogenetic analyses were used to select the most divergent sequences affiliated to these enzyme families among the 264 putative monooxygenases recovered from the ~14 million protein-coding sequences in the assembled metagenome dataset. Three-dimensional structure modeling and docking analysis suggested features useful in biotechnological applications in five metagenomic sequences, such as wide substrate range, novel substrate specificity or regioselectivity. Further analysis revealed structural features associated with psychrophilic enzymes, such as broader substrate accessibility, larger catalytic pockets or low domain interactions, suggesting that they could be applied in biooxidations at room or low temperatures, saving costs inherent to energy consumption. This work allowed the identification of putative enzyme candidates with promising features from metagenomes, providing a suitable starting point for further developments.

Immobilization of a Bacterial Cytochrome P450 Monooxygenase System on a Solid Support

AbstractBacterial cytochrome P450s (P450s), which catalyze regio‐ and stereoselective oxidations of hydrocarbons with high turnover rates, are attractive biocatalysts for fine chemical production. Enzyme immobilization is needed for cost‐effective industrial manufacturing. However, immobilization of P450s is difficult because electron‐transfer proteins are involved in catalysis and anchoring these can prevent them from functioning as shuttle molecules for carrying electrons. We studied a heterotrimeric protein‐mediated co‐immobilization of a bacterial P450, and its electron‐transfer protein and reductase. Fusion with subunits of a heterotrimeric Sulfolobus solfataricus proliferating cell nuclear antigen (PCNA) enabled immobilization of the three proteins on a solid support. The co‐immobilized enzymes catalyzed monooxygenation because the electron‐transfer protein fused to PCNA via a single peptide linker retained its electron‐transport function.

Immobilization of a Bacterial Cytochrome P450 Monooxygenase System on a Solid Support.

Bacterial cytochrome P450s (P450s), which catalyze regio- and stereoselective oxidations of hydrocarbons with high turnover rates, are attractive biocatalysts for fine chemical production. Enzyme immobilization is needed for cost-effective industrial manufacturing. However, immobilization of P450s is difficult because electron-transfer proteins are involved in catalysis and anchoring these can prevent them from functioning as shuttle molecules for carrying electrons. We studied a heterotrimeric protein-mediated co-immobilization of a bacterial P450, and its electron-transfer protein and reductase. Fusion with subunits of a heterotrimeric Sulfolobus solfataricus proliferating cell nuclear antigen (PCNA) enabled immobilization of the three proteins on a solid support. The co-immobilized enzymes catalyzed monooxygenation because the electron-transfer protein fused to PCNA via a single peptide linker retained its electron-transport function.

Enzyme-substrate complex structures of CYP154C5 shed light on its mode of highly selective steroid hydroxylation.

CYP154C5 from Nocardia farcinica is a bacterial cytochrome P450 monooxygenase active on steroid molecules. The enzyme has recently been shown to exhibit exclusive regioselectivity and stereoselectivity in the conversion of various pregnans and androstans, yielding 16α-hydroxylated steroid products. This makes the enzyme an attractive candidate for industrial application in steroid hormone synthesis. Here, crystal structures of CYP154C5 in complex with four different steroid molecules were solved at resolutions of up to 1.9 Å. These are the first reported P450 structures from the CYP154 family in complex with a substrate. The active site of CYP154C5 forms a flattened hydrophobic channel with two opposing polar regions, perfectly resembling the size and polarity distribution of the steroids and thus resulting in highly specific steroid binding with Kd values in the range 10-100 nM. Key enzyme-substrate interactions were identified that accounted for the exclusive regioselectivity and stereoselectivity of the enzyme. Additionally, comparison of the four CYP154C5-steroid structures revealed distinct structural differences, explaining the observed variations in kinetic data obtained for this P450 with the steroids pregnenolone, dehydroepiandrosterone, progesterone, androstenedione, testosterone and nandrolone. This will facilitate the generation of variants with improved activity or altered selectivity in the future by means of protein engineering.

The plmS 2 -Encoded Cytochrome P450 Monooxygenase Mediates Hydroxylation of Phoslactomycin B in Streptomyces sp. Strain HK803

Streptomyces sp. strain HK803 produces six analogues of phoslactomycin (Plm A through Plm F). With the exception of Plm B, these analogues contain a C-18 hydroxyl substituent esterified with a range of short-alkyl-chain carboxylic acids. Deletion of the plmS(2) open reading frame (ORF), showing high sequence similarity to bacterial cytochrome P450 monooxygenases (CYPs), from the Plm biosynthetic gene cluster has previously resulted in an NP1 mutant producing only Plm B (N. Palaniappan, B. S. Kim, Y. Sekiyama, H. Osada, and K. A. Reynolds, J. Biol. Chem. 278:35552-35557, 2003). Herein, we report that a complementation experiment with an NP1 derivative (NP2), using a recombinant conjugative plasmid carrying the plmS(2) ORF downstream of the ermE* constitutive promoter (pMSG1), restored production of Plm A and Plm C through Plm F. The 1.2-kbp plmS(2) ORF was also expressed efficiently as an N-terminal polyhistidine-tagged protein in Streptomyces coelicolor. The recombinant PlmS(2) converted Plm B to C-18-hydroxy Plm B (Plm G). PlmS(2) was highly specific for Plm B and unable to process a series of derivatives in which either the lactone ring was hydrolyzed or the C-9 phosphate ester was converted to C-9/C-11 phosphorinane. This biochemical analysis and complementation experiment are consistent with a proposed Plm biosynthetic pathway in which the penultimate step is hydroxylation of the cyclohexanecarboxylic acid-derived side chain of Plm B by PlmS(2) (the resulting Plm G is then esterified to provide Plm A and Plm C through Plm F). Kinetic parameters for Plm B hydroxylation by PlmS(2) (K(m) of 45.3 +/- 9.0 microM and k(cat) of 0.27 +/- 0.04 s(-1)) are consistent with this step being a rate-limiting step in the biosynthetic pathway. The penultimate pathway intermediate Plm G has less antifungal activity than Plm A through Plm F and is not observed in fermentations of either the wild-type strain or NP2/pMSG1.

Common active site architecture and binding strategy of four phenylpropanoid P450s from Arabidopsis thaliana as revealed by molecular modeling.

Despite extensive primary sequence diversity, crystal structures of several bacterial cytochrome P450 monooxygenases (P450s) and a single eukaryotic P450 indicate that these enzymes share a structural core of alpha-helices and beta-sheets and vary in the loop regions contacting individual substrates. To determine the extent to which individual structural features are conserved among divergent P450s existing in a single biosynthetic pathway, we have modeled the structures of four highly divergent P450s (CYP73A5, CYP84A1, CYP75B1, CYP98A3) in the Arabidopsis phenylpropanoid pathway synthesizing lignins, flavonoids and anthocyanins. Analysis of these models has indicated that, despite primary sequence identities as low as 13%, the structural cores and several loop regions of these P450s are highly conserved. Substrate docking indicated that all four enzymes employ a common strategy to identify their substrates in that their cinnamate-derived substrates align along helix I with their aromatic ring positioned towards the C-terminus of this helix and their aliphatic tails positioned towards the N-terminus. Further similarity was observed in the way the substrates contact the consensus P450 substrate recognition sites (SRS). Residues predicted to contact the aromatic ring region exist in SRS5, SRS6 and the C-terminal portion of SRS4 and residues contacting the distal end of each substrate exist in SRS1, SRS2 and the N-terminal portion of SRS4. Alignments of the regions contacting the aromatic ring region indicate that SRS4, SRS5 and SRS6 share higher degrees of sequence conservation than found in SRS1, SRS2 or the full-length protein.

Negative selection systems for transgenic barley (Hordeum vulgare L.): comparison of bacterial codA- and cytochrome P450 gene-mediated selection.

Efficient negative selection systems are increasingly needed for numerous applications in plant biology. In recent years various counter-selectable genes have been tested in six dicotyledonous species, whereas there are no data available for the use of negative selection markers in monocotyledonous species. In this study, we compared the applicability and reliability of two different conditional negative selection systems in transgenic barley. The bacterial codA gene encoding cytosine deaminase, which converts the non-toxic 5-fluorocytosine (5-FC) into the toxic 5-fluorouracil (5-FU), was used for in vitro selection of germinating seedlings. Development of codA-expressing seedlings was strongly inhibited by germinating the seeds in the presence of 5-FC. For selecting plants in the greenhouse, a bacterial cytochrome P450 mono-oxygenase gene, the product of which catalyses the dealkylation of a sulfonylurea compound, R7402, into its cytotoxic metabolite, was used. T1 plants expressing the selectable marker gene showed striking morphological differences from the non-transgenic plants. In experiments with both negative selectable markers, the presence or absence of the transgene, as predicted from the physiological appearance of the plants under selection, was confirmed by PCR analysis. We demonstrate that both marker genes provide tight negative selection; however, the use of the P450 gene is more amenable to large-scale screening under greenhouse or field conditions.

Nonsubstrate induction of a soluble bacterial cytochrome P-450 monooxygenase by phenobarbital and its analogs.

A soluble, cytochrome P-450-dependent fatty acid hydroxylase--epoxidase complex from Bacillus megaterium ATCC 14581 can be induced more than 100-fold by the addition of phenobarbital or one of its analogs (hexobarbital) to the growth medium. These barbiturate inducers are apparently not substrates for the enzyme nor do they activate the monooxygenase in the cell-free system. The induction efficiency of both phenobarbital and hexobarbital can be significantly increased with respect to monooxygenase activity by autoclaving the inducer in the growth medium rather than by adding it to the medium after autoclaving. Turnover numbers of about 3 000 nmoles of substrate oxygenated per min per nmole of P-450 were obtained in crude cell-free preparations obtained from maximally induced cultures. Our data indicate that products formed by heating phenobarbital or hexobarbital in the growth medium are significantly better inducers of monooxygenase activity than are the unaltered drugs.