Dataset Information

Specificity in glycosylation of multiple flagellins by the modular and cell cycle regulated glycosyltransferase FlmG.

ABSTRACT: How specificity is programmed into post-translational modification of proteins by glycosylation is poorly understood, especially for O-linked glycosylation systems. Here we reconstitute and dissect the substrate specificity underpinning the cytoplasmic O-glycosylation pathway that modifies all six flagellins, five structural and one regulatory paralog, in Caulobacter crescentus, a monopolarly flagellated alpha-proteobacterium. We characterize the biosynthetic pathway for the sialic acid-like sugar pseudaminic acid and show its requirement for flagellation, flagellin modification and efficient export. The cognate NeuB enzyme that condenses phosphoenolpyruvate with a hexose into pseudaminic acid is functionally interchangeable with other pseudaminic acid synthases. The previously unknown and cell cycle-regulated FlmG protein, a defining member of a new class of cytoplasmic O-glycosyltransferases, is required and sufficient for flagellin modification. The substrate specificity of FlmG is conferred by its N-terminal flagellin-binding domain. FlmG accumulates before the FlaF secretion chaperone, potentially timing flagellin modification, export, and assembly during the cell division cycle.

SUBMITTER: Ardissone S

PROVIDER: S-EPMC7591256 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Publications

Specificity in glycosylation of multiple flagellins by the modular and cell cycle regulated glycosyltransferase FlmG.

Ardissone Silvia S Kint Nicolas N Viollier Patrick H PH

eLife 20201027

How specificity is programmed into post-translational modification of proteins by glycosylation is poorly understood, especially for O-linked glycosylation systems. Here we reconstitute and dissect the substrate specificity underpinning the cytoplasmic O-glycosylation pathway that modifies all six flagellins, five structural and one regulatory paralog, in <i>Caulobacter crescentus</i>, a monopolarly flagellated alpha-proteobacterium. We characterize the biosynthetic pathway for the sialic acid-l ...[more]

PMID: 33108275

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Project description:Sophoricoside glycosylated derivatives, especially long-chain glycosylated sophoricosides (LCGS), have greatly improved water solubility compared with sophoricoside. Here, cyclodextrin glycosyltransferase from Paenibacillus macerans (PmCGTase) was employed for sophoricoside glycosylation. Saturation mutagenesis of alanine 156, alanine 166, glycine 173, and leucine 174 was performed due to their nonconservative properties among ?-, ?-, and ?-CGTases with different product specificities. Variants L174P, A156V/L174P, and A156V/L174P/A166Y greatly improved the product specificity for LCGS. pH significantly affected the extent of glycosylation catalyzed by the variants. Further investigations revealed that the pH-regulated mechanism for LCGS synthesis mainly depends on a disproportionation route at a lower pH (pH 4) and a cyclization-coupling route at a higher pH (pH 8) and equivalent effects of cyclization-coupling and disproportionation routes at pH 5. Whereas short-chain glycosylated sophoricosides (SCGS) are primarily produced via disproportionation of maltodextrin at pH 4 and secondary disproportionation of LCGS at pH 8. At pH 5, SCGS synthesis mainly depends on a hydrolysis route by the wild type (WT) and a secondary disproportionation route by variant A156V/L174P/A166Y. Kinetics analysis showed a decreased Km value of variant A156V/L174P/A166Y. Dynamics simulation results demonstrated that the improved LCGS specificity of the variant is possibly attributed to the enhanced affinity to long-chain substrates, which may be caused by the changes of hydrogen bond interactions at the -5, -6, and -7 subsites. Our results reveal a pH-regulated mechanism for product specificity of CGTase and provide guidance for engineering CGTase toward products with different sugar chain lengths.IMPORTANCE The low water solubility of sophoricoside seriously limits its applications in the food and pharmaceutical industries. Long-chain glycosylated sophoricosides show greatly improved water solubility. Here, the product specificity of cyclodextrin glycosyltransferase (CGTase) for long-chain glycosylated sophoricosides was significantly affected by pH. Our results reveal the pH-regulated mechanism of the glycosylated product specificity of CGTase. This work adds to our understanding of the synthesis of long-chain glycosylated sophoricosides and provides guidance for exploring related product specificity of CGTase based on pH regulation.

Dataset Information

Specificity in glycosylation of multiple flagellins by the modular and cell cycle regulated glycosyltransferase FlmG.

Publications

Specificity in glycosylation of multiple flagellins by the modular and cell cycle regulated glycosyltransferase FlmG.

Similar Datasets

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