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Cell-free enzymatic synthesis of GDP-L-fucose from mannose.

ABSTRACT: GDP-L-fucose, the key substrate for fucosyloligosaccharide biosynthesis, has been synthesized via a de novo pathway in bacteria. In the present study, genes for GDP-L-fucose biosynthesis were cloned into the expression vector pET-28a (+) to construct five E. coli strains, with recombinant enzymes being purified by using Ni-NTA chromatography. Following optimization of the 3-step reaction, Glk, ManB and ManC were added to the reaction mixture, after which Gmd and WcaG were added to overcome feedback inhibition from the end-product to produce GDP-L-fucose at 178.6 mg/l, with a yield of 14.1%. Our studies provide the basis for using cell-free enzyme production of GDP-L-fucose.

SUBMITTER: Wang W

PROVIDER: S-EPMC6536560 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Cell-free enzymatic synthesis of GDP-L-fucose from mannose.

Wang Weiyang W Zhang Fan F Wen Yanyun Y Hu Yanbo Y Yuan Ye Y Wei Min M Zhou Yifa Y

AMB Express 20190527 1

GDP-L-fucose, the key substrate for fucosyloligosaccharide biosynthesis, has been synthesized via a de novo pathway in bacteria. In the present study, genes for GDP-L-fucose biosynthesis were cloned into the expression vector pET-28a (+) to construct five E. coli strains, with recombinant enzymes being purified by using Ni-NTA chromatography. Following optimization of the 3-step reaction, Glk, ManB and ManC were added to the reaction mixture, after which Gmd and WcaG were added to overcome feedb ...[more]

PMID: 31134391

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Project description:Glycosylation of proteins is a key function of the biosynthetic-secretory pathway in the endoplasmic reticulum (ER) and Golgi apparatus. Glycosylated proteins play a crucial role in cell trafficking and signaling, cell-cell adhesion, blood-group antigenicity, and immune response. In addition, the glycosylation of proteins is an important parameter in the optimization of many glycoprotein-based drugs such as monoclonal antibodies. In vitro glycoengineering of proteins requires glycosyltransferases as well as expensive nucleotide sugars. Here, we present a designed pathway consisting of five enzymes, glucokinase (Glk), phosphomannomutase (ManB), mannose-1-phosphate-guanyltransferase (ManC), inorganic pyrophosphatase (PmPpA), and 1-domain polyphosphate kinase 2 (1D-Ppk2) expressed in E. coli for the cell-free production and regeneration of GDP-mannose from mannose and polyphosphate with catalytic amounts of GDP and ADP. It was shown that GDP-mannose is produced at various conditions, that is pH 7-8, temperature 25-35°C and co-factor concentrations of 5-20?mM MgCl2 . The maximum reaction rate of GDP-mannose achieved was 2.7??M/min at 30°C and 10?mM MgCl2 producing 566?nmol GDP-mannose after a reaction time of 240?min. With respect to the initial GDP concentration (0.8?mM) this is equivalent to a yield of 71%. Additionally, the cascade was coupled to purified, transmembrane-deleted Alg1 (ALG1?TM), the first mannosyltransferase in the ER-associated lipid-linked oligosaccharide (LLO) assembly. Thereby, in a one-pot reaction, phytanyl-PP-(GlcNAc)2 -Man1 was produced with efficient nucleotide sugar regeneration for the first time. Phytanyl-PP-(GlcNAc)2 -Man1 can serve as a substrate for the synthesis of LLO for the cell-free in vitro glycosylation of proteins. A high-performance anion exchange chromatography method with UV and conductivity detection (HPAEC-UV/CD) assay was optimized and validated to determine the enzyme kinetics. The established kinetic model enabled the optimization of the GDP-mannose regenerating cascade and can further be used to study coupling of the GDP-mannose cascade with glycosyltransferases. Overall, the study envisages a first step towards the development of a platform for the cell-free production of LLOs as precursors for in vitro glycoengineering of proteins.

Project description:Rhizobial NodZ ?1,6-fucosyltransferase (?1,6-FucT) catalyzes the transfer of the fucose (Fuc) moiety from guanosine 5'-diphosphate-?-L-fucose to the reducing end of the chitin oligosaccharide core during Nod-factor (NF) biosynthesis. NF is a key signalling molecule required for successful symbiosis with a legume host for atmospheric nitrogen fixation. To date, only two ?1,6-FucT structures have been determined, both without any donor or acceptor molecule that could highlight the structural background of the catalytic mechanism. Here, the first crystal structures of ?1,6-FucT in complex with its substrate GDP-Fuc and with GDP, which is a byproduct of the enzymatic reaction, are presented. The crystal of the complex with GDP-Fuc was obtained through soaking of native NodZ crystals with the ligand and its structure has been determined at 2.35?Å resolution. The fucose residue is exposed to solvent and is disordered. The enzyme-product complex crystal was obtained by cocrystallization with GDP and an acceptor molecule, penta-N-acetyl-L-glucosamine (penta-NAG). The structure has been determined at 1.98?Å resolution, showing that only the GDP molecule is present in the complex. In both structures the ligands are located in a cleft formed between the two domains of NodZ and extend towards the C-terminal domain, but their conformations differ significantly. The structures revealed that residues in three regions of the C-terminal domain, which are conserved among ?1,2-, ?1,6- and protein O-fucosyltransferases, are involved in interactions with the sugar-donor molecule. There is also an interaction with the side chain of Tyr45 in the N-terminal domain, which is very unusual for a GT-B-type glycosyltransferase. Only minor conformational changes of the protein backbone are observed upon ligand binding. The only exception is a movement of the loop located between strand ?C2 and helix ?C3. In addition, there is a shift of the ?C3 helix itself upon GDP-Fuc binding.

Dataset Information

Cell-free enzymatic synthesis of GDP-L-fucose from mannose.

Publications

Cell-free enzymatic synthesis of GDP-L-fucose from mannose.

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