Biochemical characterization of a novel bifunctional glycosyl-1-phosphate transferase involved in the exopolysaccharide biosynthesis

Qi, Xiaoqiang; Ma, Ming; Wang, Lan; Zhang, Yang; Jiang, Rong; Bai, Liping; Li, Yuan

doi:10.1016/j.bbrc.2015.07.140

Cited by 6 publications

(3 citation statements)

References 25 publications

(34 reference statements)

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“…Various ste genes had been functionally characterized by our lab: ste1 and ste2 for regulating exopolysaccharides production and phenotype of S sp. 139 (Bai et al 2013 ; Geng et al 2022 ); ste5 , ste7 , ste15 , and ste22 for bifunctional glycosyl-1-phosphate transferase, fucosyltransferase, glucosyltransferase, and rhamnosyltransferase, respectively (Chang et al 2009 ; Li et al 2010 ; Qi et al 2015 ; Zhang et al 2006 ); and ste8 and ste9 for polymerization and export of exopolysaccharides (Yang et al 2013 ; Zhang et al 2014 ). However, the function of the Orf2 protein encoded by the orf2 gene, situated between ste1 and ste5 (Fig.…”

Section: Introductionmentioning

confidence: 99%

Characterization and functional evidence for Orf2 of Streptomyces sp. 139 as a novel dipeptidase E

Liu,

Li,

et al. 2024

Appl Microbiol Biotechnol

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Aspartyl dipeptidase (dipeptidase E) can hydrolyze Asp-X dipeptides (where X is any amino acid), and the enzyme plays a key role in the degradation of peptides as nutrient sources. Dipeptidase E remains uncharacterized in Streptomyces. Orf2 from Streptomyces sp. 139 is located in the exopolysaccharide biosynthesis gene cluster, which may be a novel dipeptidase E with “S134-H170-D198” catalytic triad by sequence and structure comparison. Herein, recombinant Orf2 was expressed in E. coli and characterized dipeptidase E activity using the Asp-ρNA substrate. The optimal pH and temperature for Orf2 are 7.5 and 40 ℃; Vmax and Km of Orf2 are 0.0787 mM·min−1 and 1.709 mM, respectively. Orf2 exhibits significant degradation activities to Asp-Gly-Gly, Asp-Leu, Asp-His, and isoAsp-Leu and minimal activities to Asp-Pro and Asp-Ala. Orf2 contains a Ser-His-Asp catalytic triad characterized by point mutation. In addition, the Asp147 residue of Orf2 is also proven to be critical for the enzyme’s activity through molecular docking and point mutation. Transcriptome analysis reveals the upregulation of genes associated with ribosomes, amino acid biosynthesis, and aminoacyl-tRNA biosynthesis in the orf2 mutant strain. Compared with the orf2 mutant strain and WT, the yield of crude polysaccharide does not change significantly. However, crude polysaccharides from the orf2 mutant strain exhibit a wider range of molecular weight distribution. The results indicate that the Orf2 links nutrient stress to secondary metabolism as a novel dipeptidase E. Key points • A novel dipeptidase E with a Ser-His-Asp catalytic triad was characterized from Streptomyces sp. 139. • Orf2 was involved in peptide metabolism both in vitro and in vivo. • Orf2 linked nutrient stress to mycelia formation and secondary metabolism in Streptomyces.

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Section: Introductionmentioning

confidence: 99%

Characterization and functional evidence for Orf2 of Streptomyces sp. 139 as a novel dipeptidase E

Liu,

Li,

et al. 2024

Appl Microbiol Biotechnol

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“…Glycosyl 1-phosphates are key intermediates in carbohydrate primary metabolism and are utilised by microorganisms to form polyphosphate architectures that constitute keys parts of their extracellular capsule and cell walls [1][2][3][4][5]. They serve as precursors to sugar-nucleotides [6,7], the sugar-donor components utilised by glycosyltransferases in the assembly of oligosaccharides and glycans and have played a key role in the development of glycosylated natural-product-based therapeutics [8].…”

Section: Introductionmentioning

confidence: 99%

6R/S-deutero-α-d-mannopyranoside 1-phosphate

Ahmadipour

Miller

2019

Molbank

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6R/S-deutero-α-d-mannopyranoside 1-phosphate was synthesised from a C6 aldehydic mannose thioglycoside donor in four steps. Using NaBD4 as the reductant, isotopic enrichment at C6 was achieved and the resultant C6-deuterated material was converted through to the glycosyl 1-phosphate using a protection/glycosylation/deprotection sequence. The product was fully characterised by 1H, 13C, 31P and 2D NMR, alongside MS analysis.

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“…For some PGTs, high-throughput assays using fluorescence have also been developed, however, these approaches require modification of the glycosyl phosphate donor, which is only feasible with PGTs such as MraY, where the fluorophore can be incorporated distal to the reacting groups 16 . Alternatively, multienzyme-based analyses, which couple UMP release to NAD + generation, have been employed 17 .…”

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confidence: 99%

A Rapid and Efficient Luminescence-based Method for Assaying Phosphoglycosyltransferase Enzymes

Das

Walvoort

Lukose

et al. 2016

Sci Rep

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Phosphoglycosyltransferases (PGTs) are families of integral membrane proteins with intriguingly diverse architectures. These enzymes function to initiate many important biosynthetic pathways including those leading to peptidoglycan, N-linked glycoproteins and lipopolysaccharide O-antigen. In spite of tremendous efforts, characterization of these enzymes remains a challenge not only due to the inherent difficulties associated with the purification of integral membrane proteins but also due to the limited availability of convenient assays. Current PGT assays include radioactivity-based methods, which rely on liquid-liquid or solid-liquid extractions, multienzyme systems linked to lactate dehydrogenase and NAD+ generation, and HPLC-based approaches, all of which may suffer from low sensitivity and low throughput. Herein, we present the validation of a new luminescence-based assay (UMP-Glo) for measuring activities of PGT enzymes. This assay measures UMP, the by-product of PGT reactions, in a sensitive and quantitative manner by measuring the luminescence output in a discontinuous coupled assay system. The assay is rapid and robust in nature, and also compatible with microtiter plate formats. Activity and kinetic parameters of PglC, a PGT from Campylobacter jejuni, were quickly established using this assay. The efficacy of the assay was further corroborated using two different PGTs; PglC from Helicobacter pullorum and WecA from Thermatoga maritima.

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Biochemical characterization of a novel bifunctional glycosyl-1-phosphate transferase involved in the exopolysaccharide biosynthesis

Cited by 6 publications

References 25 publications

Characterization and functional evidence for Orf2 of Streptomyces sp. 139 as a novel dipeptidase E

Characterization and functional evidence for Orf2 of Streptomyces sp. 139 as a novel dipeptidase E

6R/S-deutero-α-d-mannopyranoside 1-phosphate

A Rapid and Efficient Luminescence-based Method for Assaying Phosphoglycosyltransferase Enzymes

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