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 plan t CYP73A orthologues from other species were isolated from a cDNA library d erived from mRNAs expressed in elicitor-treated suspension-cultured cells. These clones appear to code for a full-length 1554 bp open reading frame wi th 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 M-r of 59 229 and a pI of 8.8. It contains the conserved c ysteine haem-binding site found in all cytochrome P450s. The protein encode d 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 a ssociated with a novel regulatory mechanism. Sequence similarity is suffici ent for it to be classified as CYP73A15, although it is the least similar m ember of this family classified so far. The cDNA was expressed in yeast. Su ccessful expression of cinnamate 4-hydroxylase activity required removal of the intron. High-level expression also required modification of the N-term inus to that of CYP73A1. Yeast did not process the intron at all and the le ader sequence for A15 was not as compatible as that of A1. The mRNA for CYP 73A15 was shown to be rapidly induced by elicitor treatment of suspension-c ultured cells of French bean but induction was more transient than that of phenylalanine ammonia-lyase (PAL). In contrast, induction in cells undergoi ng xylogenesis was much more coordinate with PAL. The cloned cDNA may repre sent a cinnamate 4-hydroxylase isoform, whose expression is more related to differentiation than the responses to stress in which the majority of CYP7 3As cloned so far are involved.