|PENG, HUI - Nanjing Agricultural University|
|WHITAKER, BRUCE - Retired ARS Employee|
|SHANGGUAN, LINFEI - Nanjing Agricultural University|
|FANG, JINGGUI - Nanjing Agricultural University|
Submitted to: Biomed Central (BMC) Plant Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/1/2016
Publication Date: 9/8/2016
Citation: Peng, H., Yang, T., Whitaker, B.D., Shangguan, L. 2016. Calcium/calmodulin alleviates substrate inhibition in a strawberry UDP-glucosyltransferase involved in fruit anthocyanin biosynthesis. Biomed Central (BMC) Plant Biology. 16:197. doi:10.1186/s12870-016-0888-z.
Interpretive Summary: Anthocyanins are a class of important secondary metabolites, function as colorful pigments to protect plants against pathogenic and insect attack. Anthocyanins are also antioxidants which potentially protect human against oxidative stress mediated diseases such as cancer and inflammation. UDP-glucosyltransferase (UGT) is a key enzyme during anthocyanin biosynthesis by catalyzing glucosylation of anthocyanins so as to increase their solubility and accumulation. Previously it has been shown that preharvest spray of calcium chloride enhances anthocyanin accumulation in strawberry fruit by stimulating the expression of anthocynin structural genes including the fruit specific FvUGT1. The objective of this study is to investigate the kinetic dynamics of the recombinant FvUGT1. The results showed that FvUGT1 was subjected to feed-forward substrate inhibition and feedback product inhibition. In addition, calcium/calmodulin specifically interacted to FvUGT1, and significantly increased the enzyme activity. These results will benefit the scientists and breeders to use the knowledge for controlling the anthocyanin biosynthesis in fruits and vegetables so as to improve fruit quality.
Technical Abstract: UDP-glucosyltransferase (UGT) is a key enzyme during anthocyanin biosynthesis by catalyzing glucosylation of anthocyanins so as to increase their solubility and accumulation. Previously it has been shown that preharvest spray of calcium chloride enhances anthocyanin accumulation in strawberry fruit by stimulating the expression of anthocynin structural genes including the fruit specific FvUGT1. To further understand the regulatory mechanism of anthocyanin biosynthesis, the kinetic characterization of the recombinant FvUGT1 was performed on glucosylation of pelargonidin, a major anthocyanidin in strawberry fruit. At fixed pelargonidin concentration, FvUGT1 catalyzed the transfer of UDP-glucose basically following Michaelis – Menten kinetics. In contrast, at fixed UDP-glucose, pelargonidin exhibited a clear substrate inhibition above 150 micro M in a partial uncompetitive mode. FvUGT1 was also subjected to feedback inhibition when the end product pelargonidin-3-O-glucoside was over 100 micro M. In addition, calcium/calmodulin specifically bound to the site overlapping with the interdomain linker of FvUGT1, and significantly relieved the substrate inhibition. In the presence of 0.1 micro M calmodulin, the Vm was increased by 71.4%. These results suggest that FvUGT1 activity is allosterically regulated by both sugar acceptor substrate and glycoside product, and calcium/calmodulin can enhance anthocyanin accumulation by relieving the substrate inhibition on FvUGT1.