Molecular investigations of plant cytochrome P450

Purification of plant cyt P450s has been difficult, due to their general low abundance in plant tissues and instability during purification. This has meant that only a few have been directly purified from plant tissue to date. Thus, conventional methods for studying their catalytic activity, such as cloning procedures involving the use of antibodies, or the generation of oligonucleotide probes from amino acid sequence, have proven of limited success. An alternative molecular approach was therefore adopted to study cyt P450s from Zea mays and Arabidopsis. For Z. mays, degenerate PCR primers directed to two conserved regions of sequence were used to amplify a cyt P450 fragment with greatest homology to the CYP71 family of plant cyt P450s. Despite extensive screening of phage Z. mays leaf, safener-treated root and seedling cDNA libraries, a full-length clone was not isolated using this fragment as a probe. Genomic southern analysis confirmed that a gene corresponding to the cyt P450 fragment was part of the Z. mays genome. Northern analysis and reverse transcriptase-polymerase chain reaction indicated that it represented a transcript of low abundance, was present in shoot and root tissues and was constitutive with respect to age and treatments known to induce cyt P450s capable of herbicide metabolism. For Arabidopsis, an Expressed Sequence Tag (EST) from the Michigan State University EST programme, previously identified as a potential full-length cyt P450, was obtained for further analysis in an effort to assign function. Detailed sequence analysis confirmed it was a full-length cyt P450 of the CYP71 family, and it was designated CYP71B7. Northern analysis revealed it was expressed most strongly in rosette leaves and was also present in roots, leaves, siliques and flowers. Southern analysis indicated that it represented a single copy gene in the Arabidopsis genome. CYP71B7 was successfully expressed in Saccharomyces cerevisiae and biochemical analysis revealed it was active in ethoxycoumarin O-deethylation. 7-ethoxycoumarin is a general artificial substrate of cyt P450s. Several natural compounds were identified which inhibited this activity, and thus are candidate physiological substrates of CYP71B7