Investigating Potentially Key Residues Which Imparts the Substrate and Regiospecifi city of aFlavonol-Specifi c 3-O-Glucosyltransferase from Grapefruit

Most naturally-occurring fl avonoids are found in glucosylated form. Glucosyltransferases (GTs) are enzymes that catalyze the transfer of glucose from a high energy sugar donor to an acceptor molecule. Citrus paradisi fl avonol-specifi c glucosyltransferase (Cp-F3-O-GT) is recognized for its rigid substrate and regiospecifi city. In this work, homology modeling, site-directed mutagenesis, and biochemical analyses of the recombinant mutant Cp-F3-O-GT proteins were used to investigate potential amino acid residues that might be responsible for the enzymes strict regiospecifi city while also investigating its substrate specifi city. The single point mutations of three amino acid residues within the grapefruit F3-O-GT identifi ed through sequence alignment and homology modeling were performed. Analyses of the enzyme activity of the recombinant mutant F3-O-GT proteins revealed that the single point mutations of serine 20 to leucine (S20L) and proline 297 to phenylalanine (P297F) rendered the recombinant enzymes inactive with fl avonol substrates at 6% and 12% respectively relative to wild-type. However, the mutation of glycine 392 to glutamate (G392E) remained active and glucosylated the fl avonol acceptors quercein (Km app= 11 μM; Vmax = 5.7 pKat/μg) relative to the wild-type (Km app= 93 μM; Vmax = 41.7 pKat/μg), and kaempferol (Km app= 7 μM; Vmax = 3.8 pKat/μg) relative to the wild-type (Km app = 39 μM; Vmax = 4.2 pKat/ μg). The mutant enzyme also did not show broadened acceptor substrate specifi city as it also favored fl avonols as the preferred acceptor substrate. The optimum pH of the mutant enzyme was 8.0 similar to the wild-type F3-O-GT. Activity of the mutant enzyme was stimulated by NaCl and KCl, but inhibited by Cu2+, Zn2+, Fe2+ as well as UDP with an apparent Ki of 10μM. Product identifi cation to determine glucosylation position is being investigated for a possible change in regiospecifi city