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

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.


FvUGT1 kinetics toward UDP-Glc under the fixed pelargonidin (150 μM). a. Data were fitted to the Michaelis-Menten equation. b. The Lineweaver-Burk plot showing the linear relationship between 1/[pelargonidin] and 1/V. Each point represents the mean velocity +/−SD from triplicate determinations
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Fig3: FvUGT1 kinetics toward UDP-Glc under the fixed pelargonidin (150 μM). a. Data were fitted to the Michaelis-Menten equation. b. The Lineweaver-Burk plot showing the linear relationship between 1/[pelargonidin] and 1/V. Each point represents the mean velocity +/−SD from triplicate determinations

Mentions: To investigate the enzyme kinetics of FvUGT1, we selected the indirect Glycosyltransferase Activity assay rather than HPLC-based approach since the former approach were high-throughput yet had the comparable results to those by the latter approach [33–35]. Before determining the kinetic parameters of FvUGT1, we optimized the reaction conditions including reaction temperature and reaction time. The recombinant FvUGT1 exhibited higher activity at 37 °C than 30 °C. For the time course assay, we found that product formation showed a linear positive correlation with incubation time in the range of 10–30 min, indicating that the initial velocities were consistent within 30 min (data not shown). Thus, all initial velocity experiments were performed at 37 °C for 30 min. At the fixed concentration of pelargonidin chloride (150 μM), the resulting plot for UDP-Glc (Fig. 3a) followed the classical Michaelis-Menten kinetics (Eq. 1), as evidenced by the linear Lineweaver-Burk plot (Fig. 3b). The Vmax and Km for UDP-Glc were around 12.7 nmol · s−1 · mg−1 and 201.8 μM, respectively. However, when adding more than 150 μM pelargonidin, the Vmax declined significantly although the plot still followed Michaelis-Menten kinetics (data not shown). Thus high pelargonidin could have an inhibitory effect on the enzyme activity.Fig. 3


Calcium/calmodulin alleviates substrate inhibition in a strawberry UDP-glucosyltransferase involved in fruit anthocyanin biosynthesis
FvUGT1 kinetics toward UDP-Glc under the fixed pelargonidin (150 μM). a. Data were fitted to the Michaelis-Menten equation. b. The Lineweaver-Burk plot showing the linear relationship between 1/[pelargonidin] and 1/V. Each point represents the mean velocity +/−SD from triplicate determinations
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5017016&req=5

Fig3: FvUGT1 kinetics toward UDP-Glc under the fixed pelargonidin (150 μM). a. Data were fitted to the Michaelis-Menten equation. b. The Lineweaver-Burk plot showing the linear relationship between 1/[pelargonidin] and 1/V. Each point represents the mean velocity +/−SD from triplicate determinations
Mentions: To investigate the enzyme kinetics of FvUGT1, we selected the indirect Glycosyltransferase Activity assay rather than HPLC-based approach since the former approach were high-throughput yet had the comparable results to those by the latter approach [33–35]. Before determining the kinetic parameters of FvUGT1, we optimized the reaction conditions including reaction temperature and reaction time. The recombinant FvUGT1 exhibited higher activity at 37 °C than 30 °C. For the time course assay, we found that product formation showed a linear positive correlation with incubation time in the range of 10–30 min, indicating that the initial velocities were consistent within 30 min (data not shown). Thus, all initial velocity experiments were performed at 37 °C for 30 min. At the fixed concentration of pelargonidin chloride (150 μM), the resulting plot for UDP-Glc (Fig. 3a) followed the classical Michaelis-Menten kinetics (Eq. 1), as evidenced by the linear Lineweaver-Burk plot (Fig. 3b). The Vmax and Km for UDP-Glc were around 12.7 nmol · s−1 · mg−1 and 201.8 μM, respectively. However, when adding more than 150 μM pelargonidin, the Vmax declined significantly although the plot still followed Michaelis-Menten kinetics (data not shown). Thus high pelargonidin could have an inhibitory effect on the enzyme activity.Fig. 3

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.