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.