Catalytic Properties of the Plant Cytochrome P450 CYP73 Expressed in Yeast

Abstract

have been investigated. Microsomes from transformed yeast catalysed trans-cinnamate hydroxylation with high efficiency. CYP73 was highly specific for its natural substrate, and did not catalyse oxygenation of p-coumarate, benzoate, ferulate, naringenin or furanocoumarins. No metabolism of terpenoids or fatty acids, known substrates of plant P450s, was observed. CYP73 however demethylated the natural coumarin herniarin into umbelliferone. In addition, it was shown to oxygenate five xenobiotics and mechanism-based inactivators, including the herbicide chlorotoluron. All substrates of CYP73 were small planar aromatic molecules. Comparison of the kinetic parameters of CYP73 for its various substrates showed that, as expected, cinnamate was by far the best substrate of this P450. The physiological and toxicological significance of these observations are discussed.Cytochromes P450 form a large superfamily of several hundreds to several thousands of hemoproteins, involved in oxygen activation and oxygen tranfer into lipophilic molecules. They all share some sequence identity related to their common catalytic properties, i.e. heme and oxygen binding, electron transfer and oxygen activation [l]. Differences in their primary sequences usually reflect variations in specificity for substrates oxygenated. These differences may concern more than 80% of the amino acid sequence. However, the modification of a single amino acid residue can be sufficient to completely alter the substrate specificity of the enzyme 121.Full-length amino acid sequences presently available for plant P450s share less than 30% identity. This almost certainly implies great differences in the structures of their substrate binding sites. However, no relation between the structure of the enzymes and their catalytic activities has yet been established. CYP73 is the first DNA sequence coding for a plant P450 with an identified physiological function to have been isolated 131. It catalyses the 4-hydroxylation of trunscinnamic acid into p-coumaric acid. This hydroxylation is ductase (4-hydroxylating) (EC 1.14.13.11).the second reaction in the phenylpropanoid pathway. Cinnamate 4-hydroxylase (CA4H) is thus an obligatory step for the biosynthesis of lignin, a major component of the earth's total biomass. It is involved in the formation of most of the phenylpropanoid derivatives, essential for plant development, pigmentation and defense against both ultraviolet light and pathogens. Recent data [4-61 suggest that CA4H plays a central role in the regulation of the phenylpropanoid pathway. We, therefore, decided to establish more precisely which molecules are transformed by the enzyme or interfere with its catalytic activity. The determination of a population of substrates was also intended to provide information for analysis of the structurelfunction relationship of this plant P450. We devised an optimized system for the expression of CYP73 in Saccharomyces cerevisae [7]. This system provided yeast microsomes for which the only detectable P450 was CYP73. It routine...

CYP98A3 from Arabidopsis thaliana Is a 3′-Hydroxylase of Phenolic Esters, a Missing Link in the Phenylpropanoid Pathway

“…CYP98A3 Is a 3Ј-Hydroxylase of p-Coumaric Acid EstersSubstrate specificity was investigated using recombinant CYP98A3 in yeast microsomes coexpressed with the A. thaliana P450 reductase ATR1. Free phenylpropanoids were shown to be the substrates of the P450 enzymes involved in 4-and 5-hydroxylations of the aromatic ring of the C 3 -C 6 structure for lignin biosynthesis: cinnamic acid in the case of CYP73 enzymes (8,21) and coniferyladehyde, coniferyl alcohol, and, to a lesser extend, ferulic acid in the case of CYP84 enzymes (6,22). The first tests were thus performed with free p-coumarate, p-coumaraldehyde, and p-coumaroyl alcohol, which did not induce the shift in the CYP98A3 heme iron spin state that would be expected upon binding of a P450 ligand (Type I ligand binding spectrum) (19,23) or any trace of conversion into a more oxygenated molecule in the presence of NADPH and CYP98A3.…”

“…Although previously foreseen by Heller and Kü hnl (7), this new development is rather unexpected and raises a new degree of complexity and additional gridding level in the already complex lignin biosynthesis pathway. [3-14 C]cinnamate (Isotopchim, Ganagobie, France) using microsomes from recombinant yeast expressing CYP73A1 (8). The 4-and 3-isomers of p-coumaroylshikimic acid were generated by heating a solution of the trans-5-O-isomer in 0.1 M sodium phosphate buffer (pH 7.4) for 1 h at 90°C, and the cis-isomer of trans-5-O-p-coumaroylshikimic acid was obtained by irradiation for 10 min at 254 nm (9).…”

Xenobiotic Biotransformation in Unicellular Green Algae (Involvement of Cytochrome P450 in the Activation and Selectivity of the Pyridazinone Pro-Herbicide Metflurazon)

“…Anti-SU1 (CYP105Al) and anti-SU2 (CYP105Bl) are from bacterial antigens of Streptomyces griseolus (OKeefe et al, 1988;Omer et al, 1990). It is noteworthy that, except for AOS, P450s originally detected by these antibodies have already been documented to be participants in the biotransformation of distinct xenobiotics (OKeefe et al, 1988;OKeefe and Leto, 1989;Pierrel et al, 1994).…”

Arabidopsis CYP98A3 Mediating Aromatic 3-Hydroxylation. Developmental Regulation of the Gene, and Expression in Yeast

“…Of the three hydroxylases implicated in ring substitutions, C4H was the first to be studied and the best characterized cytochrome P450 monooxygenase (CYP) from plants (Pierrel et al, 1994;Urban et al, 1994;Chapple, 1998;Blount et al, 2000). It is encoded by a single gene in Arabidopsis and is designated as CYP73A5 (http://drnelson.utmem.edu/Cytochrome P450.html).…”