High throughput mass spectrometry-based characterisation of <i>Arabidopsis thaliana</i> group H glycosyltransferases

Akere, Aishat Mojisola; Liu, Qian; Wu, Shuai; Hou, Bing-Kai; Yang, Min

doi:10.1039/c8ra03947j

Cited by 4 publications

(7 citation statements)

References 30 publications

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“…UDP-Glc was the most commonly used donor for the screened UGTs and is in agreement with previous research which highlighted its frequent use among plant UGTs as the favored donor compound [50]. UGTs 76E1, 76E2, 76D1, and 76E5 were able to use UDP-Glc as their substrate, consistent with our previous finding [28]. Unlike others, 76E4 showed no activity with any of the donor sugars screened.…”

Section: Donor Specificitysupporting

confidence: 92%

“…They were then verified via Sanger DNA sequencing (Source Bioscience Ltd). The bacterial cells were grown as previously reported [ 28 ]. The GST-tagged recombinant protein was purified by affinity chromatography (columns) and quantified by the Bradford assay.…”

Section: Methodsmentioning

confidence: 99%

“…The supernatant was then analyzed with LC–MS. Glycosylated products were identified by their molecular mass, and these target compounds were subsequently fragmented using MS/MS for confirmation [ 28 ].…”

Section: Methodsmentioning

confidence: 99%

“…The reaction mixture was incubated at 37°C for 15 h, terminated with acetonitrile and centrifuged to remove proteins. The supernatant was then analyzed with LC-MS. Glycosylated products were identified by their molecular mass, and these target compounds were subsequently fragmented using MS/MS for confirmation [28].…”

Section: In Vitro Ugt Reaction Assaymentioning

confidence: 99%

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Structure-based enzyme engineering improves donor-substrate recognition of Arabidopsis thaliana glycosyltransferases

Akere

Chen

Liu

et al. 2020

Biochemical Journal

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Glycosylation of secondary metabolites involves plant UDP-dependent glycosyltransferases (UGTs). UGTs have shown promise as catalysts in the synthesis of glycosides for medical treatment. However, limited understanding at the molecular level due to insufficient biochemical and structural information has hindered potential applications of most of these UGTs. In the absence of experimental crystal structures, we employed advanced molecular modelling and simulations in conjunction with biochemical characterisation to design a workflow to study five Group H Arabidopsis thaliana (76E1, 76E2, 76E4, 76E5, 76D1) UGTs. Based on our rational structural manipulation and analysis, we identified key amino acids (P129 in 76D1; D374 in 76E2; K275 in 76E4), which when mutated improved donor-substrate recognition than wildtype UGTs. Molecular dynamics simulations and deep learning analysis identified structural differences, which drive substrate preferences. The design of these UGTs with broader substrate specificity may play important role in biotechnological and industrial applications. These findings can also serve as basis to study other plant UGTs and thereby advancing UGT enzyme engineering.

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Section: Donor Specificitysupporting

confidence: 92%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: In Vitro Ugt Reaction Assaymentioning

confidence: 99%

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Structure-based enzyme engineering improves donor-substrate recognition of Arabidopsis thaliana glycosyltransferases

Akere

Chen

Liu

et al. 2020

Biochemical Journal

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“…These characteristic fragmentation patterns associated with the glycosylation sites were consistent with the previous studies. 29,30 Then, the possible glycosylation sites of flavonoid mono-O-glycosides were recommended according to characteristic fragmentation pattern.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Novel Method for Comprehensive Annotation of Plant Glycosides Based on Untargeted LC-HRMS/MS Metabolomics

et al. 2022

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Glycosides are a large family of secondary metabolites in plants, which play a critical role in plant growth and development. Due to the complexity and diversity in structures and the limited availability of authentic standards, comprehensive annotation of the glycosides remains a great challenge. In this study, using maize as an example, a deep annotation method of glycosides was proposed based on untargeted liquid chromatography–high-resolution tandem mass spectrometry metabolomics analysis. First, knowledge-based in silico aglycone and glycosyl/acyl-glycosyl libraries were built. A total of 1240 known and potential aglycones from databases and literature were recorded. Next, the MS parameters beneficial to aglycone ion-rich MS/MS were explored using 1782 high-resolution MS/MS spectra of glycosides from the MassBank of North America (MoNA) and confirmed by 52 authentic glycoside standards. Then, screening rules for aglycon ions in MS/MS were recommended. Glycoside candidates were further filtered by MS/MS-based chemical classification and MS/MS similarity of aglycon–glycoside pairs. Finally, the glycosylation sites of flavonoid mono-O-glycosides were recommended by characteristic fragmentation patterns. The developed method was validated using glycosides and nonglycosides from the MoNA library. The annotation accuracy rates were 96.8, 94.9, and 98.0% in negative ion mode (ESI–), positive ion mode (ESI+), and the combined ESI– & ESI+, respectively. The annotation specificity was 99.6% (ESI–), 99.6% (ESI+), and 99.2% (ESI– & ESI+). A total of 274 glycosides (including 34 acyl-glycosides) were tentatively annotated in maize by the developed method. The method enables effective and reliable annotation for plant glycosides.

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Metabolic engineering for the synthesis of steviol glycosides: current status and future prospects

Zhou

Gong

Liu

et al. 2021

Appl Microbiol Biotechnol

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High throughput mass spectrometry-based characterisation of Arabidopsis thaliana group H glycosyltransferases

Abstract: We cloned and characterised four group H glycosyltransferases by studying their substrate specificities and kinetics. Sequence alignment and site-directed mutagenesis studies showed that serine is a crucial residue for UDPGlcNAc and UDPGal activity.

Cited by 4 publications

References 30 publications

Structure-based enzyme engineering improves donor-substrate recognition of Arabidopsis thaliana glycosyltransferases

Structure-based enzyme engineering improves donor-substrate recognition of Arabidopsis thaliana glycosyltransferases

Novel Method for Comprehensive Annotation of Plant Glycosides Based on Untargeted LC-HRMS/MS Metabolomics

Metabolic engineering for the synthesis of steviol glycosides: current status and future prospects

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