A Novel Glucosylation Reaction on Anthocyanins Catalyzed by Acyl-Glucose–Dependent Glucosyltransferase in the Petals of Carnation and Delphinium  

Abstract

Glucosylation of anthocyanin in carnations (Dianthus caryophyllus) and delphiniums (Delphinium grandiflorum) involves novel sugar donors, aromatic acyl-glucoses, in a reaction catalyzed by the enzymes acyl-glucose-dependent anthocyanin 5 (7)-O-glucosyltransferase (AA5GT and AA7GT). The AA5GT enzyme was purified from carnation petals, and cDNAs encoding carnation Dc AA5GT and the delphinium homolog Dg AA7GT were isolated. Recombinant Dc AA5GT and Dg AA7GT proteins showed AA5GT and AA7GT activities in vitro. Although expression of Dc AA5GT in developing carnation petals was highest at early stages, AA5GT activity and anthocyanin accumulation continued to increase during later stages. Neither Dc AA5GT expression nor AA5GT activity was observed in the petals of mutant carnations; these petals accumulated anthocyanin lacking the glucosyl moiety at the 5 position. Transient expression of Dc AA5GT in petal cells of mutant carnations is expected to result in the transfer of a glucose moiety to the 5 position of anthocyanin. The amino acid sequences of Dc AA5GT and Dg AA7GT showed high similarity to glycoside hydrolase family 1 proteins, which typically act as b-glycosidases. A phylogenetic analysis of the amino acid sequences suggested that other plant species are likely to have similar acylglucose-dependent glucosyltransferases.

Structural Determinants Allowing Transferase Activity in SENSITIVE TO FREEZING 2, Classified as a Family I Glycosyl Hydrolase

“…In doing so, a strategy was developed to yield higher confidence models. This strategy is applicable to other (␤/␣) 8 barrel protein homology modeling efforts, and likely it will be applicable to other protein structures in which the enzyme core is conserved, although loops associated with substrate selectivity are more variable in sequence. The SFR2 model surprisingly yields a catalytic site identical in sequence and similar in architecture to that of GH1s with hydrolase but no transferase activity.…”

“…Structurally, they adopt a (␤/␣) 8 or "␣/␤" barrel protein fold, also known as a triose-phosphate isomerase barrel, with loop regions conferring substrate specificity and modulating activity (6). Identified in many organisms, GH1 family proteins have always been found to have hydrolase activity, until the discovery of SFR2 and two additional GH1s described as transferases in plants as follows: Oryza sativa (rice) Os9BGlu31 synthesizes phytohormone glycoconjugates (7), and Dianthus caryophyllus (carnation) AA5GT glucosylates anthocyanin (8). The naturally occurring structural changes required to mechanistically convert a hydrolase into a structurally similar transferase are unknown (9).…”

“…These search engines were used because each has a unique ranking system for candidates. Top candidates identified by each program were then compared manually with preference given to candidates with higher sequence identity to SFR2, particularly in GH1 motifs and catalytic residues, and the best coverage of the entire (␤/␣) 8 barrel. This method identified Sulfolobus solfataricus PDB entry 1UWT, a ␤-glycosidase in complex with D-galactohydroximo-1,5-lactam (17) and Rauvolfia serpentina PDB entry 4A3Y, a raucaffricine glucosidase (18) as the best structural templates for the barrel region of the protein.…”

“…They were then manually compiled into a multiple sequence alignment such that the majority of the (␤/␣) 8 barrel GH1 motifs were aligned with 1UWT or 4A3Y, although loops of SFR2 were aligned with corresponding loops from the best loop templates.…”

The Roles of a Flavone-6-Hydroxylase and 7-O-Demethylation in the Flavone Biosynthetic Network of Sweet Basil

“…Their structural variation, reflected by more than 9,000 compounds reported to date, is achieved through "decorative" steps, such as glycosylations, acylations, prenylations, methylations, hydroxylations, and combinations of the above (2). Because chemodiversity correlates with a diversity of function, specific strategies and mechanisms of chemical structural modification are of great interest (3,4). Numerous bioactivities have been reported for various flavonoids, turning them into attractive targets for bioengineering experiments (5,6).…”