Cloning and sequence analysis of a mutation-type cinnamate 4-hydroxylase gene from L. var. DC.

Abstract

A 2431-bp full-length cinnamate 4-hydroxylase gene, BoC4H, was cloned from Brassica oleracea L. var. acephala DC.. It contains 2 introns. Its mRNA is 1715 bp, encoding a deduced 481-amino-acid polypeptide with wide homologies to C4Hs from other plants. It possesses cytochrome P450 conserved domains and motifs such as the haem-iron binding motif, the E-R-R triad, the T-containing binding pocket motif and the hinge motif necessary for optimal orientation of the enzyme. It also has most of the canonical C4H/CYP73A5-featured substrate-recognition sites (SRSs) and active site residues. However, owing to a single-base deletion at C₂₂₄₂ and subsequent frame shift within the 3′ coding region as compared with C4H genes from Arabidopsis thaliana and other plants, BoC4H shows a 36-aa deletion/variation at its C-terminus and the SRS6 motif together with active site residues therein are absent. Thus BoC4H may be of no function or low activity. BoC4H is a membrane protein and is probably associated with the endoplasmic reticulum. Its secondary structure is dominated by alpha helices and random coils. The Swiss-Model could not predict its tertiary structure. B. oleracea contains a C4H gene family with at least 5 members.

Keywords

Brassica oleracea L. var. acephala DC. / cloning, cinnamate 4-hydroxylase (C4H) / mutation / sequence analysis