Dataset Information

A Parsimonious Mechanism of Sugar Dehydration by Human GDP-Mannose-4,6-dehydratase.

ABSTRACT: Biosynthesis of 6-deoxy sugars, including l-fucose, involves a mechanistically complex, enzymatic 4,6-dehydration of hexose nucleotide precursors as the first committed step. Here, we determined pre- and postcatalytic complex structures of the human GDP-mannose 4,6-dehydratase at atomic resolution. These structures together with results of molecular dynamics simulation and biochemical characterization of wildtype and mutant enzymes reveal elusive mechanistic details of water elimination from GDP-mannose C5? and C6?, coupled to NADP-mediated hydride transfer from C4? to C6?. We show that concerted acid-base catalysis from only two active-site groups, Tyr179 and Glu157, promotes a syn 1,4-elimination from an enol (not an enolate) intermediate. We also show that the overall multistep catalytic reaction involves the fewest position changes of enzyme and substrate groups and that it proceeds under conserved exploitation of the basic (minimal) catalytic machinery of short-chain dehydrogenase/reductases.

SUBMITTER: Pfeiffer M

PROVIDER: S-EPMC6454399 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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A Parsimonious Mechanism of Sugar Dehydration by Human GDP-Mannose-4,6-dehydratase.

Pfeiffer Martin M Johansson Catrine C Krojer Tobias T Kavanagh Kathryn L KL Oppermann Udo U Nidetzky Bernd B

ACS catalysis 20190301 4

Biosynthesis of 6-deoxy sugars, including l-fucose, involves a mechanistically complex, enzymatic 4,6-dehydration of hexose nucleotide precursors as the first committed step. Here, we determined pre- and postcatalytic complex structures of the human GDP-mannose 4,6-dehydratase at atomic resolution. These structures together with results of molecular dynamics simulation and biochemical characterization of wildtype and mutant enzymes reveal elusive mechanistic details of water elimination from GDP ...[more]

PMID: 30984471

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Project description:BACKGROUND: Antibody-dependent cellular cytotoxicity (ADCC) is greatly enhanced by the absence of the core fucose of oligosaccharides attached to the Fc, and is closely related to the clinical efficacy of anticancer activity in humans in vivo. Unfortunately, all licensed therapeutic antibodies and almost all currently-developed therapeutic antibodies are heavily fucosylated and fail to optimize ADCC, which leads to a large dose requirement at a very high cost for the administration of antibody therapy to cancer patients. In this study, we explored the possibility of converting already-established antibody-producing cells to cells that produce antibodies fully lacking core fucosylation in order to facilitate the rapid development of next-generation therapeutic antibodies. RESULTS: Firstly, loss-of-function analyses using small interfering RNAs (siRNAs) against the three key genes involved in oligosaccharide fucose modification, i.e. alpha1,6-fucosyltransferase (FUT8), GDP-mannose 4,6-dehydratase (GMD), and GDP-fucose transporter (GFT), revealed that single-gene knockdown of each target was insufficient to completely defucosylate the products in antibody-producing cells, even though the most effective siRNA (>90% depression of the target mRNA) was employed. Interestingly, beyond our expectations, synergistic effects of FUT8 and GMD siRNAs on the reduction in fucosylation were observed, but not when these were used in combination with GFT siRNA. Secondly, we successfully developed an effective short hairpin siRNA tandem expression vector that facilitated the double knockdown of FUT8 and GMD, and we converted antibody-producing Chinese hamster ovary (CHO) cells to fully non-fucosylated antibody producers within two months, and with high converting frequency. Finally, the stable manufacture of fully non-fucosylated antibodies with enhanced ADCC was confirmed using the converted cells in serum-free fed-batch culture. CONCLUSION: Our results suggest that FUT8 and GMD collaborate synergistically in the process of intracellular oligosaccharide fucosylation. We also demonstrated that double knockdown of FUT8 and GMD in antibody-producing cells could serve as a new strategy for producing next-generation therapeutic antibodies fully lacking core fucosylation and with enhanced ADCC. This approach offers tremendous cost- and time-sparing advantages for the development of next-generation therapeutic antibodies.

Dataset Information

A Parsimonious Mechanism of Sugar Dehydration by Human GDP-Mannose-4,6-dehydratase.

Publications

A Parsimonious Mechanism of Sugar Dehydration by Human GDP-Mannose-4,6-dehydratase.

Similar Datasets

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