Donor Assists Acceptor Binding and Catalysis of Human α1,6-Fucosyltransferase

α1,6-Core-fucosyltransferase (FUT8) is a vital enzyme in mammalian physiological and pathophysiological processes such as tumorigenesis and progress of, among others, non-small cell lung cancer and colon carcinoma. It was also shown that therapeutic antibodies have a dramatically higher efficacy if...

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Veröffentlicht in:ACS chemical biology 2013-08, Vol.8 (8), p.1830-1840
Hauptverfasser: Kötzler, Miriam P, Blank, Simon, Bantleon, Frank I, Wienke, Martin, Spillner, Edzard, Meyer, Bernd
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container_end_page 1840
container_issue 8
container_start_page 1830
container_title ACS chemical biology
container_volume 8
creator Kötzler, Miriam P
Blank, Simon
Bantleon, Frank I
Wienke, Martin
Spillner, Edzard
Meyer, Bernd
description α1,6-Core-fucosyltransferase (FUT8) is a vital enzyme in mammalian physiological and pathophysiological processes such as tumorigenesis and progress of, among others, non-small cell lung cancer and colon carcinoma. It was also shown that therapeutic antibodies have a dramatically higher efficacy if the α1,6-fucosyl residue is absent. However, specific and potent inhibitors for FUT8 and related enzymes are lacking. Hence, it is crucial to elucidate the structural basis of acceptor binding and the catalytic mechanism. We present here the first structural model of FUT8 in complex with its acceptor and donor molecules. An unusually large acceptor, i.e., a hexasaccharide from the core of N-glycans, is required as minimal structure. Acceptor substrate binding of FUT8 is being dissected experimentally by STD NMR and SPR and theoretically by molecular dynamics simulations. The acceptor binding site forms an unusually large and shallow binding site. Binding of the acceptor to the enzyme is much faster and stronger if the donor is present. This is due to strong hydrogen bonding between O6 of the proximal N-acetylglucosamine and an oxygen atom of the β-phosphate of GDP-fucose. Therefore, we propose an ordered Bi Bi mechanism for FUT8 where the donor molecule binds first. No specific amino acid is present that could act as base during catalysis. Our results indicate a donor-assisted mechanism, where an oxygen of the β-phosphate deprotonates the acceptor. Knowledge of the mechanism of FUT8 is now being used for rational design of targeted inhibitors to address metastasis and prognosis of carcinomas.
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It was also shown that therapeutic antibodies have a dramatically higher efficacy if the α1,6-fucosyl residue is absent. However, specific and potent inhibitors for FUT8 and related enzymes are lacking. Hence, it is crucial to elucidate the structural basis of acceptor binding and the catalytic mechanism. We present here the first structural model of FUT8 in complex with its acceptor and donor molecules. An unusually large acceptor, i.e., a hexasaccharide from the core of N-glycans, is required as minimal structure. Acceptor substrate binding of FUT8 is being dissected experimentally by STD NMR and SPR and theoretically by molecular dynamics simulations. The acceptor binding site forms an unusually large and shallow binding site. Binding of the acceptor to the enzyme is much faster and stronger if the donor is present. This is due to strong hydrogen bonding between O6 of the proximal N-acetylglucosamine and an oxygen atom of the β-phosphate of GDP-fucose. 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subjects Binding Sites
Carbohydrate Sequence
Catalysis
Fucosyltransferases - chemistry
Fucosyltransferases - metabolism
Humans
Models, Molecular
Molecular Dynamics Simulation
Peptides - chemistry
Peptides - genetics
title Donor Assists Acceptor Binding and Catalysis of Human α1,6-Fucosyltransferase
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