Calcium/calmodulin alleviates substrate inhibition in a strawberry UDP-glucosyltransferase involved in fruit anthocyanin biosynthesis.
UDP-glucosyltransferase (UGT) is a key enzyme for anthocyanin biosynthesis, which by catalyzing glycosylation of anthocyanidins increases their solubility and accumulation in plants. Previously we showed that pre-harvest spray of CaCl2 enhanced anthocyanin accumulation in strawberry fruit by stimulating the expression of anthocyanin structural genes including a fruit specific FvUGT1.



To further understand the regulation of anthocyanin biosynthesis, we conducted kinetic analysis of recombinant FvUGT1 on glycosylation of pelargonidin, the major anthocyanidin in strawberry fruit. At the fixed pelargonidin concentration, FvUGT1 catalyzed the sugar transfer from UDP-glucose basically following Michaelis-Menten kinetics. By contrast, at the fixed UDP-glucose concentration, pelargonidin over 150 μM exhibited marked partial substrate inhibition in an uncompetitive mode. These results suggest that the sugar acceptor at high concentration inhibits FvUGT1 activity by binding to another site in addition to the catalytic site. Furthermore, calcium/calmodulin specifically bound FvUGT1 at a site partially overlapping with the interdomain linker, and significantly relieved the substrate inhibition. In the presence of 0.1 and 0.5 μM calmodulin, V max was increased by 71.4 and 327 %, respectively.



FvUGT1 activity is inhibited by anthocyanidin, the sugar acceptor substrate, and calcium/calmodulin binding to FvUGT1 enhances anthocyanin accumulation via alleviation of this substrate inhibition.
Agricultural Research Service of U.S. Department of Agriculture, From the Food Quality Laboratory, Beltsville Agricultural Research Center, Beltsville, MD, 20705, USA.|Horticulture & Landscape College, Hunan Agricultural University, Changsha, Hunan, 410128, China.
Hui
H
Peng
Agricultural Research Service of U.S. Department of Agriculture, From the Food Quality Laboratory, Beltsville Agricultural Research Center, Beltsville, MD, 20705, USA. tianbao.yang@ars.usda.gov.
Tianbao
T
Yang
Agricultural Research Service of U.S. Department of Agriculture, From the Food Quality Laboratory, Beltsville Agricultural Research Center, Beltsville, MD, 20705, USA.
Bruce D
BD
Whitaker
Agricultural Research Service of U.S. Department of Agriculture, From the Food Quality Laboratory, Beltsville Agricultural Research Center, Beltsville, MD, 20705, USA.|College of Horticulture, Nanjing Agricultural University, Nanjing, Jiangsu, 210095, China.
Lingfei
L
Shangguan
College of Horticulture, Nanjing Agricultural University, Nanjing, Jiangsu, 210095, China.
Jinggui
J
Fang
D000595:Amino Acid Sequence; D000872:Anthocyanins / Q000235:genetics*; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D031985:Fragaria / Q000201:enzymology* / Q000235:genetics*; D005638:Fruit / Q000201:enzymology; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006031:Glycosylation; D007700:Kinetics; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment
D000872:Anthocyanins; D002147:Calmodulin; D010940:Plant Proteins; D005964:Glucosyltransferases; D002118:Calcium
Calcium signaling; Enzyme kinetics; Fragaria vesca; Pelargonidin; UGT