The terminal O-acetyltransferase involved in vindoline biosynthesis defines a new class of proteins responsible for coenzyme A-dependent acyl transfer.
The gene encoding acetyl CoA:deacetylvindoline 4-O-acetyltransferase (DAT) (EC 2.3.1.107) which catalyzes the last step in vindoline biosynthesis was isolated and characterized. The genomic clone encoded a 50 kDa polypeptide containing the sequences of nine tryptic fragments derived from the purified DAT heterodimer. However, cleavage of DAT protein to yield a heterodimer appears to be an artifact of the protein purification procedure, since the size of the protein (50 kDa) cross-reacting with anti-DAT antibody in seedlings and in leaves of various ages also corresponds to the size of the active recombinant enzyme. Studies with the intact plant and with developing seedlings showed that induction of DAT mRNA, protein accumulation and enzyme activity occurred preferentially in vindoline producing tissues such as leaves and cotyledons of light-treated etiolated seedlings. The ORF of DAT showed significant sequence identity to 19 other plant genes, whose biochemical functions were mostly unknown. The Mr of approximately 50 kDa, a HXXXDG triad, and a DFGWGKP consensus sequence are highly conserved among the 20 plant genes and these criteria may be useful to identify this type of acyltransferase. The involvement of some of these genes in epicuticular wax biosynthesis, fruit-ripening and in benzoyltransfer reactions indicates that the plant kingdom contains a superfamily of multifunctional acyltransferases which operate by a reaction mechanism related to the ancient chloramphenicol O-acetyltransferase and dihydrolipoyl acetyltransferase class of enzymes.
St-Pierre
B
B
Département de Sciences Biologiques, Université de Montréal, Québec, Canada.
Laflamme
P
P
Alarco
A M
AM
De Luca
V
V
D000123:Acetyltransferases / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003065:Coenzyme A / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D008969:Molecular Sequence Data; D010946:Plants, Medicinal / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D014747:Vinblastine / Q000031:analogs & derivatives* / Q000096:biosynthesis
D017931:DNA Primers; D018744:DNA, Plant; C009667:vindoline; D014747:Vinblastine; D000123:Acetyltransferases; C064442:acetylcoenzyme A-deacetylvindoline 4-O-acetyltransferase; D003065:Coenzyme A