Increasing expression of P450 and P450-reductase proteins from monocots in heterologous systems.

Monocotyledonous crop plants are usually more resistant to herbicides than grass weeds and most dicots. Their resistance to herbicides is mediated in many cases by P450 oxygenases. Monocots thus constitute an appealing source of P450 enzymes for manipulating herbicide resistance and recombinant forms of the major xenobiotic metabolizing mooxygenases are potential tools for the optimization of new active molecules. We report here the isolation and functional characterization of the first P450 and P450 reductase coding sequences from wheat. The first attempts at expressing these cDNAs in yeast and tobacco led to levels of protein, which were extremely low, often not even detectable. The wheat P450 cDNAs were efficiently transcribed, but no protein or activity was found. Wheat coding sequences, like those of other monocots, are characterized by a high GC content and by a related strong bias of codon usage, different from that observed in yeast or dicots. Complete recoding of genes being costly, the reengineering their 5'-end using a single PCR megaprimer designed to comply with codon usage of the host was attempted. It was sufficient to relieve translation inhibition and to obtain good levels of protein expression. The same strategy also resulted in a dramatic increase in protein expression in tobacco. A basis for the success of such a partial recoding strategy, much easier and cheaper than complete recoding of the cDNA, is proposed.

Novel characteristics and regulation of a divergent cinnamate 4-hydroxylase (CYP73A15) from French bean: engineering expression in yeast.

cDNAs showing high sequence similarity (>70%) over large stretches to plant CYP73A orthologues from other species were isolated from a cDNA library derived from mRNAs expressed in elicitor-treated suspension-cultured cells. These clones appear to code for a full-length 1554 bp open reading frame with a 78 bp 5'-untranslated region and a 140 bp 3'-untranslated region. The open reading frame, determined by sequence similarity, codes for a protein with a predicted Mr of 59229 and a pI of 8.8. It contains the conserved cysteine haem-binding site found in all cytochrome P450s. The protein encoded by this cDNA diverges however from other CYP73As in its N- and C-terminus and in four domains internally, so that overall sequence similarity is in the range 58-66%. Many clones contained an identical intron, which may be associated with a novel regulatory mechanism. Sequence similarity is sufficient for it to be classified as CYP73A15, although it is the least similar member of this family classified so far. The cDNA was expressed in yeast. Successful expression of cinnamate 4-hydroxylase activity required removal of the intron. High-level expression also required modification of the N-terminus to that of CYP73A1. Yeast did not process the intron at all and the leader sequence for A15 was not as compatible as that of A1. The mRNA for CYP73A15 was shown to be rapidly induced by elicitor treatment of suspension-cultured cells of French bean but induction was more transient than that of phenylalanine ammonia-lyase (PAL). In contrast, induction in cells undergoing xylogenesis was much more coordinate with PAL. The cloned cDNA may represent a cinnamate 4-hydroxylase isoform, whose expression is more related to differentiation than the responses to stress in which the majority of CYP73As cloned so far are involved.

Role of unusual amino acid residues in the proximal and distal heme regions of a plant P450, CYP73A1.

CYP73A1 is a typical plant P450 in terms of its function and primary sequence. The enzyme catalyzes the 4-hydroxylation of trans-cinnamic acid, the first oxidative step in the phenylpropanoid pathway. Its primary protein sequence exhibits some particular landmarks which are characteristic of plant P450 enzymes. The most interesting is a proline residue (Pro448), very unusual in animal P450s, just C-terminal to the invariant heme-binding cysteine. To determine the role of this proline, we substituted it with valine, isoleucine, or phenylalanine, residues found in animal P450s, using site-directed mutagenesis. Expression of the wild type and mutants in yeast indicated that replacement of Pro448 led to disruption of the heme-protein interaction, loss of catalytic activity, and either impaired expression or destabilization of the apoprotein. Pro448 is thus essential for the correct insertion of heme in the apoprotein. Another typical feature of CYP73A proteins is the presence of an alanine-alanine motif (Ala306-Ala307) on the presumed N-terminal edge of the cleft in the central part of the I helix. This cleft faces the iron on the distal side of the heme and is proposed to be essential for catalysis. Substitution of each or both Ala306 and Ala307 residues with glycines showed that they are critical for the stability of the protein and influence the positioning of the substrate in the active site. Results are discussed with reference to the structural X-ray data that are available for bacterial P450 proteins.

The chemically inducible plant cytochrome P450 CYP76B1 actively metabolizes phenylureas and other xenobiotics

Cytochrome P450s (P450s) constitute one of the major classes of enzymes that are responsible for detoxification of exogenous molecules both in animals and plants. On the basis of its inducibility by exogenous chemicals, we recently isolated a new plant P450, CYP76B1, from Jerusalem artichoke (Helianthus tuberosus) and showed that it was capable of dealkylating a model xenobiotic compound, 7-ethoxycoumarin. In the present paper we show that CYP76B1 is more strongly induced by foreign compounds than other P450s isolated from the same plant, and metabolizes with high efficiency a wide range of xenobiotics, including alkoxycoumarins, alkoxyresorufins, and several herbicides of the class of phenylureas. CYP76B1 catalyzes the double N-dealkylation of phenylureas with turnover rates comparable to those reported for physiological substrates and produces nonphytotoxic compounds. Potential uses for CYP76B1 thus include control of herbicide tolerance and selectivity, as well as soil and groundwater bioremediation.

Cloning and functional expression in yeast of a cDNA coding for an obtusifoliol 14alpha-demethylase (CYP51) in wheat.

Screening of a wheat cDNA library with an heterologous CYP81B1 probe from Helianthus tuberosus led to the isolation of a partial cDNA coding a protein with all the characteristics of a typical P450 with high homology (32-39% identity) to the fungal and mammalian CYP51s. Extensive screening of several wheat cDNA libraries isolated a longer cDNA (W516) coding a peptide of 453 amino acids. Alignment of W516 with other P450 sequences revealed that it was missing a segment corresponding to the N-terminal membrane anchor of the protein. The corresponding segment from the yeast lanosterol 14alpha-demethylase was linked to the partial wheat cDNA and the chimera expressed in Saccharomyces cerevisiae. Compared to microsomes from control yeasts, membranes of yeast expressing the chimera catalysed 14alpha-demethylation of obtusifoliol with an increased efficiency relative to lanosterol demethylase activity. W516 is thus a plant member of the most ancient and conserved P450 family, CYP51.

Design of fluorescent substrates and potent inhibitors of CYP73As, P450s that catalyze 4-hydroxylation of cinnamic acid in higher plants.

CYP73As are the major functional cytochromes P450 in higher plants. Several of them have been shown to catalyze the 4-hydroxylation of cinnamic acid, the first oxidative step in the synthesis of lignin, flavonoids, coumarins, and other phenylpropanoids. The coding sequence for CYP73A1, the enzyme from Helianthus tuberosus, has been isolated and expressed in yeast. Previous studies indicate that the yeast-expressed enzyme is capable of metabolizing cinnamic acid and several small, planar molecules but with low efficiency. Using this we further examined how CYP73A1 could bind and metabolize a set of possible alternate substrates. We show here that naphthalenes, quinolines, and indoles substituted with an aldehyde, a carboxylic, or a sulfonic acid group make good ligands and substrates for CYP73A1. The best ligands are hydroxynaphthoic acids, which show higher affinity than cinnamate. Naphthalene, 2-naphthol, and molecules with two-carbon side chains, such as natural and synthetic auxins, are not substrates of this enzyme. Methyl-2-naphthoate and 2-hydroxy-1-naphthoic acid are strong ligands of CYP73A1 but are not metabolized. Uncoupling and low spin conversion induced by these compounds suggest that their positioning in the heme pocket is inadequate for catalysis. These compounds can act as potent inhibitors of the second step of the phenylpropanoid pathway, the first described so far. The molecule which most closely mimics cinnamic acid, 2-naphthoic acid, is metabolized with a catalytic turnover and efficiency similar to those measured with the physiological substrate. Using this compound we designed a fluorometric assay to measure the catalytic activity of CYP73As. This assay was then used to monitor the CYP73As activity in microsomes from transgenic yeast and several plant species.