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Functional diversification of two UGT80 enzymes required for steryl glucoside synthesis in Arabidopsis.

Stucky DF, Arpin JC, Schrick K - J. Exp. Bot. (2014)

Bottom Line: A third related enzyme, which seems specific to the plant lineage, is encoded by UGT713B1/At5g24750.Although the results demonstrate specific activities for UGT80A2 and UGT80B1, a role for UGT713B1 in SG synthesis was not supported.This study extends our knowledge of UGT80 enzymes and provides evidence for specialized functions for distinct classes of SG and ASG molecules in plants.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology, Kansas State University, Manhattan, KS 66506-4901, USA Molecular, Cellular and Developmental Biology, Kansas State University, Manhattan, KS 66506-4901, USA.

No MeSH data available.


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In vitro substrate specificity of UGT80A2 and UGT80B1 enzymes. (A–C) Yeast microsomes expressing the empty vector control, UGT80A2, UGT80B1, or UGT713B1 were incubated with sterol and UDP-glucose substrates. Reaction products were analysed by ESI-MS/MS and normalized signals per sample are shown. Error bars indicate standard deviations for n=3. Glucosylated sterols were detected for UGT80A2 and UGT80B1, but not for UGT713B1. Signals from the empty vector control ranged from 0.001 to 0.043 (Supplementary Table S6). Fold changes over the negative control were calculated using an average value of 0.020 for the control, and each fold-change represents a significant increase in activity over the control (P≤0.05). (A) UGT80A2 and UGT80B1 expression resulted in glucosylated cholesterol when using UDP-glucose as the substrate. (B) UGT80A2 and UGT80B1 displayed high activity with endogenous ergosterol for the UDP-glucose substrate. (C) UGT80A2 and UGT80B1 formed sitosteryl, stigmasteryl, campesteryl and brassicasteryl glucosides utilizing UDP-glucose in combination with a sterol mixture containing the corresponding sterols.
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Figure 6: In vitro substrate specificity of UGT80A2 and UGT80B1 enzymes. (A–C) Yeast microsomes expressing the empty vector control, UGT80A2, UGT80B1, or UGT713B1 were incubated with sterol and UDP-glucose substrates. Reaction products were analysed by ESI-MS/MS and normalized signals per sample are shown. Error bars indicate standard deviations for n=3. Glucosylated sterols were detected for UGT80A2 and UGT80B1, but not for UGT713B1. Signals from the empty vector control ranged from 0.001 to 0.043 (Supplementary Table S6). Fold changes over the negative control were calculated using an average value of 0.020 for the control, and each fold-change represents a significant increase in activity over the control (P≤0.05). (A) UGT80A2 and UGT80B1 expression resulted in glucosylated cholesterol when using UDP-glucose as the substrate. (B) UGT80A2 and UGT80B1 displayed high activity with endogenous ergosterol for the UDP-glucose substrate. (C) UGT80A2 and UGT80B1 formed sitosteryl, stigmasteryl, campesteryl and brassicasteryl glucosides utilizing UDP-glucose in combination with a sterol mixture containing the corresponding sterols.

Mentions: The UGT80A2, UGT80B1, and UGT713B1 enzymes were heterologously expressed in a yeast (S. cerevisiae) strain deleted for UGT51/YLR189C. Yeast UGT51, also known as UGT51A1, codes for an enzyme that glycosylates ergosterol (Warnecke et al., 1999), and deletion thereof eliminates SG production. Yeast microsomal fractions individually expressing the Arabidopsis UGT80A2, UGT80B1, and UGT713B1 enzymes were incubated with various sterol substrates and UDP-glucose or UDP-glucuronide substrates to test for the production of SG. Subsequently, a direct infusion ESI-MS/MS approach was used to quantify SG products from the reactions (Schrick et al., 2012b) (Fig. 6; Supplementary Tables S2, S3, and S6). The data indicate that both the UGT80A2 and UGT80B1 enzymes are able to glycosylate all sterols tested using UDP-glucose as a substrate. However, no detectable enzyme activity was observed with any of the sterol substrates with UDP-glucuronide as a substrate (Tables S3; S6).


Functional diversification of two UGT80 enzymes required for steryl glucoside synthesis in Arabidopsis.

Stucky DF, Arpin JC, Schrick K - J. Exp. Bot. (2014)

In vitro substrate specificity of UGT80A2 and UGT80B1 enzymes. (A–C) Yeast microsomes expressing the empty vector control, UGT80A2, UGT80B1, or UGT713B1 were incubated with sterol and UDP-glucose substrates. Reaction products were analysed by ESI-MS/MS and normalized signals per sample are shown. Error bars indicate standard deviations for n=3. Glucosylated sterols were detected for UGT80A2 and UGT80B1, but not for UGT713B1. Signals from the empty vector control ranged from 0.001 to 0.043 (Supplementary Table S6). Fold changes over the negative control were calculated using an average value of 0.020 for the control, and each fold-change represents a significant increase in activity over the control (P≤0.05). (A) UGT80A2 and UGT80B1 expression resulted in glucosylated cholesterol when using UDP-glucose as the substrate. (B) UGT80A2 and UGT80B1 displayed high activity with endogenous ergosterol for the UDP-glucose substrate. (C) UGT80A2 and UGT80B1 formed sitosteryl, stigmasteryl, campesteryl and brassicasteryl glucosides utilizing UDP-glucose in combination with a sterol mixture containing the corresponding sterols.
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Related In: Results  -  Collection

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Figure 6: In vitro substrate specificity of UGT80A2 and UGT80B1 enzymes. (A–C) Yeast microsomes expressing the empty vector control, UGT80A2, UGT80B1, or UGT713B1 were incubated with sterol and UDP-glucose substrates. Reaction products were analysed by ESI-MS/MS and normalized signals per sample are shown. Error bars indicate standard deviations for n=3. Glucosylated sterols were detected for UGT80A2 and UGT80B1, but not for UGT713B1. Signals from the empty vector control ranged from 0.001 to 0.043 (Supplementary Table S6). Fold changes over the negative control were calculated using an average value of 0.020 for the control, and each fold-change represents a significant increase in activity over the control (P≤0.05). (A) UGT80A2 and UGT80B1 expression resulted in glucosylated cholesterol when using UDP-glucose as the substrate. (B) UGT80A2 and UGT80B1 displayed high activity with endogenous ergosterol for the UDP-glucose substrate. (C) UGT80A2 and UGT80B1 formed sitosteryl, stigmasteryl, campesteryl and brassicasteryl glucosides utilizing UDP-glucose in combination with a sterol mixture containing the corresponding sterols.
Mentions: The UGT80A2, UGT80B1, and UGT713B1 enzymes were heterologously expressed in a yeast (S. cerevisiae) strain deleted for UGT51/YLR189C. Yeast UGT51, also known as UGT51A1, codes for an enzyme that glycosylates ergosterol (Warnecke et al., 1999), and deletion thereof eliminates SG production. Yeast microsomal fractions individually expressing the Arabidopsis UGT80A2, UGT80B1, and UGT713B1 enzymes were incubated with various sterol substrates and UDP-glucose or UDP-glucuronide substrates to test for the production of SG. Subsequently, a direct infusion ESI-MS/MS approach was used to quantify SG products from the reactions (Schrick et al., 2012b) (Fig. 6; Supplementary Tables S2, S3, and S6). The data indicate that both the UGT80A2 and UGT80B1 enzymes are able to glycosylate all sterols tested using UDP-glucose as a substrate. However, no detectable enzyme activity was observed with any of the sterol substrates with UDP-glucuronide as a substrate (Tables S3; S6).

Bottom Line: A third related enzyme, which seems specific to the plant lineage, is encoded by UGT713B1/At5g24750.Although the results demonstrate specific activities for UGT80A2 and UGT80B1, a role for UGT713B1 in SG synthesis was not supported.This study extends our knowledge of UGT80 enzymes and provides evidence for specialized functions for distinct classes of SG and ASG molecules in plants.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology, Kansas State University, Manhattan, KS 66506-4901, USA Molecular, Cellular and Developmental Biology, Kansas State University, Manhattan, KS 66506-4901, USA.

No MeSH data available.


Related in: MedlinePlus