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Calcium/calmodulin alleviates substrate inhibition in a strawberry UDP-glucosyltransferase involved in fruit anthocyanin biosynthesis

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ABSTRACT

Background: 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.

Results: 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, Vmax was increased by 71.4 and 327 %, respectively.

Conclusions: 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.

Electronic supplementary material: The online version of this article (doi:10.1186/s12870-016-0888-z) contains supplementary material, which is available to authorized users.

No MeSH data available.


FvUGT1 kinetics showing that calmodulin can alleviate pelargonidin inhibition. a. Data were fitted to the partial uncompetitive inhibition model (Eq. 2). b. The Lineweaver-Burk plot is shown to illustrate that calmodulin did not fully relieve the inhibition kinetics. CaM, calmodulin. Each point represents the mean velocity +/−SD from triplicate determinations (●, 0 μM Calmodulin; ■, 0.1 μM Calmodulin; ♦, 0.5 μM Calmodulin; ▲, 2.5 μM Calmodulin). The concentration of UDP-Glc in the reactions was kept fixed (5 mM)
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Fig6: FvUGT1 kinetics showing that calmodulin can alleviate pelargonidin inhibition. a. Data were fitted to the partial uncompetitive inhibition model (Eq. 2). b. The Lineweaver-Burk plot is shown to illustrate that calmodulin did not fully relieve the inhibition kinetics. CaM, calmodulin. Each point represents the mean velocity +/−SD from triplicate determinations (●, 0 μM Calmodulin; ■, 0.1 μM Calmodulin; ♦, 0.5 μM Calmodulin; ▲, 2.5 μM Calmodulin). The concentration of UDP-Glc in the reactions was kept fixed (5 mM)

Mentions: To determine the effect of calmodulin binding on enzyme activity, FvUGT1 enzymatic kinetics was further analyzed in the presence of calcium/calmodulin. Since calcium was required for coupling phosphatase activity assay, we were only able to investigate the effect of different calmodulin concentrations on FvUGT1 activity. Adding calmodulin significantly increased the enzyme activity (Fig. 6a), although the plot of velocity vs. pelargonidin concentration still exhibited a substrate inhibition mode, as evidenced by the non-linear Lineweaver-Burk plots (Fig. 6b). To obtain the best fit, the x value was still kept as 3, suggesting that calmodulin-binding to FvUGT1 does not affect pelargonidin binding affinity to the additional site. In the presence of 0.1, 0.5, and 2.5 μM calmodulin, the apparent Vmax was increased by 71.4 %, 227 % and 246 %, respectively. These results suggest that calmodulin-binding to FvUGT1 partially relieved the substrate inhibition.Fig. 6


Calcium/calmodulin alleviates substrate inhibition in a strawberry UDP-glucosyltransferase involved in fruit anthocyanin biosynthesis
FvUGT1 kinetics showing that calmodulin can alleviate pelargonidin inhibition. a. Data were fitted to the partial uncompetitive inhibition model (Eq. 2). b. The Lineweaver-Burk plot is shown to illustrate that calmodulin did not fully relieve the inhibition kinetics. CaM, calmodulin. Each point represents the mean velocity +/−SD from triplicate determinations (●, 0 μM Calmodulin; ■, 0.1 μM Calmodulin; ♦, 0.5 μM Calmodulin; ▲, 2.5 μM Calmodulin). The concentration of UDP-Glc in the reactions was kept fixed (5 mM)
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Fig6: FvUGT1 kinetics showing that calmodulin can alleviate pelargonidin inhibition. a. Data were fitted to the partial uncompetitive inhibition model (Eq. 2). b. The Lineweaver-Burk plot is shown to illustrate that calmodulin did not fully relieve the inhibition kinetics. CaM, calmodulin. Each point represents the mean velocity +/−SD from triplicate determinations (●, 0 μM Calmodulin; ■, 0.1 μM Calmodulin; ♦, 0.5 μM Calmodulin; ▲, 2.5 μM Calmodulin). The concentration of UDP-Glc in the reactions was kept fixed (5 mM)
Mentions: To determine the effect of calmodulin binding on enzyme activity, FvUGT1 enzymatic kinetics was further analyzed in the presence of calcium/calmodulin. Since calcium was required for coupling phosphatase activity assay, we were only able to investigate the effect of different calmodulin concentrations on FvUGT1 activity. Adding calmodulin significantly increased the enzyme activity (Fig. 6a), although the plot of velocity vs. pelargonidin concentration still exhibited a substrate inhibition mode, as evidenced by the non-linear Lineweaver-Burk plots (Fig. 6b). To obtain the best fit, the x value was still kept as 3, suggesting that calmodulin-binding to FvUGT1 does not affect pelargonidin binding affinity to the additional site. In the presence of 0.1, 0.5, and 2.5 μM calmodulin, the apparent Vmax was increased by 71.4 %, 227 % and 246 %, respectively. These results suggest that calmodulin-binding to FvUGT1 partially relieved the substrate inhibition.Fig. 6

View Article: PubMed Central - PubMed

ABSTRACT

Background: 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.

Results: 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, Vmax was increased by 71.4 and 327 %, respectively.

Conclusions: 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.

Electronic supplementary material: The online version of this article (doi:10.1186/s12870-016-0888-z) contains supplementary material, which is available to authorized users.

No MeSH data available.