MOLECULAR-CLONING AND CHARACTERIZATION OF DESACETOXYVINDOLINE-4-HYDROXYLASE, A 2-OXOGLUTARATE DEPENDENT DIOXYGENASE INVOLVED IN THE BIOSYNTHESIS OF VINDOLINE IN CATHARANTHUS-ROSEUS (L) DON,G

A 2-oxoglutarate-dependent dioxygenase (EC 1.14.11.11) which catalyzes the 4-hydroxylation of desacetoxyvindoline was purified to homogeneit y. Three oligopeptides isolated from a tryptic digest of the purified protein were microsequenced and one oligopeptide showed significant ho mology to hyoscyamine GP-hydroxylase from Hyoscyamus niger. A 36-mer d egenerate oligonucleotide based on this peptide sequence was used to s creen a Catharanthus rosens cDNA library and three clones, cD4H-1 to - 3, were isolated. Although none of the three clones were full-length, the open reading frame on each clone encoded a putative protein contai ning the sequence of all three peptides. Primer extension analysis sug gested that cD4H-3, the longest cDNA clone, was missing 156 bp at the 5' end of the clone and sequencing of the genomic clone, gD4H-8, confi rmed these results. Southern blot analysis suggested that d4h is prese nt as a single-copy gene in C. roseus which is a diploid plant, and th e significant differences in the sequence of the 3'-UTR between cD4H-1 and -3 suggest that they represent dimorphic alleles of the same hydr oxylase. The identity of the clone was further confirmed when extracts of transformed Escherichia coli expressed D4H enzyme activity. The D4 H clone encoded a putative protein of 401 amino acids with a calculate d molecular mass of 45.5 kDa and the amino acid sequence showed a high degree of similarity with those of a growing family of 2-oxoglutarate -dependent dioxygenases of plant and fungal origin. The similarity was not restricted to the dioxygenase protein sequences but was also exte nded to the gene structure and organization since the 205 and 1720 bp introns of d4h were inserted around the same highly conserved amino ac id consensus sequences as those for e8 protein, hyoscyamine-6 beta-hyd roxylase and ethylene-forming enzyme. These results provide further su pport that a common ancestral gene is responsible far the appearance o f this family of dioxygenases. Hydroxylase assays and RNA blot hybridi zation studies showed that enzyme activity followed closely the levels of d4h transcripts, occurring predominantly in young leaves and in mu ch lower levels in stems and fruits. In contrast, etiolated seedlings which contained considerable levels of d4h transcripts had almost unde tectable hydroxylase activity, whereas exposure of seedlings to light resulted in a rapid increase of enzyme activity without a significant further increase in d4h transcripts over those detected in dark-grown seedlings. These results suggest that the activating effect of light m ay occur at a point downstream of transcription which remains to be el ucidated.