ISOLATION AND CHARACTERIZATION OF A CDNA FROM CELL-SUSPENSION CULTURES OF VANILLA-PLANIFOLIA ENCODING 4-COUMARATE - COENZYME-A LIGASE

cDNA clones encoding 4-coumarate-CoA ligase (4CL) (EC 6.2.1.12), a key enzyme of phenylpropanoid metabolism, were isolated from a lambda-Zap II cDNA library constructed from mRNA of a kinetin-treated cell suspen sion culture of Vanilla planifolia Andr. Complete nucleotide sequence of a full length cDNA clone (Vp4cl-9) was determined. It contains a 16 62 bp open reading frame coding for 553 amino acid residues, a 14 bp 5 ' end sequence and a 221 bp 3' end non-coding sequence. The cDNA seque nce and the deduced amino acid sequence of Vp4cl-9 were compared to ot her plant 4CLs. The nucleotide sequence showed 76% to 60% homology, wh ile the deduced amino acid sequence showed 83% to 60% identity with so me deletions and insertions in the N-terminal part of the enzyme. A co nsensus sequence based on thirteen plant 4CLs has been generated. A hi gh degree of conservation is observed in the C-terminal half of the en zymes. Computer-based sequence analysis revealed three putative ATP bi nding sites at residue position 197-207 (YSSGTTGLPKG), 395-401 (GEICIR G) and 429-431 (DIG). The latter sequence is contained in a domain str ucture of the protein with a predicted beta-sheet: alpha-helix: beta-s heet fold. Furthermore, two putative glycosylation sites: NIS at posit ion 38-40 and NIT at 499-501 were found. Detection of 4CL activity in extracts of Escherichia coli XL-1 Blue cells transformed with a pBlues cript phagemid construct containing Vp4cl-9, confirmed that Vp4cl-9 en codes 4CL. Other Vp4CL cDNA clones were also partially sequenced. The cDNA sequences and deduced amino acid sequences of these clones are al l identical to Vp4cl-9. However, the results from Southern analysis, s uggest that there are at least two 4CL genes in the Vanilla genome. A phylogenetic analysis using 19 4CL deduced amino acid sequences showed that the Vanilla 4CL is most related to the tobacco and potato 4CL en zymes.