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

Abstract

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.

Structural basis of substrate recognition and catalysis by fucosyltransferase 8

“…This dimer could be observed in the previously determined apo structure of FUT8 21 , yet the structure was reported on as a monomer. Other publications have reported that HsFUT8 is a monomer in solution, based on size exclusion chromatography experiments 33 , and this has influenced the way in which molecular dynamics and docking simulations of FUT8 have been conducted 22,23 , potentially compromising their conclusions. To address this inconsistency in the literature, we performed SEC-SAXS on FUT8 ( Figure 4B,C), which conclusively demonstrated that FUT8 exists as a dimer in solution.…”

“…To some degree, drug discovery efforts are impeded by a limited structural understanding of this enzyme and the mechanism it employs to perform core fucosylation. The only reported FUT8 structure possesses no bound ligands, 21 and our only insights into donor and acceptor substrate binding come from STD-NMR, molecular dynamics and docking studies 22,23 . To gain a thorough understanding of how FUT8 recognises both its donor and acceptor substrates to catalyse core fucosylation, we revisited the structural biology of FUT8.…”

Structural basis of substrate recognition and catalysis by fucosyltransferase 8

“…This dimer could be observed in the previously determined apo structure of FUT8 21 , yet the structure was reported on as a monomer. Other publications have reported that HsFUT8 is a monomer in solution, based on size exclusion chromatography experiments 33 , and this has influenced the way in which molecular dynamic and docking simulations of FUT8 have been conducted 22, 23 , potentially compromising their conclusions. To address this inconsistency in the literature, we performed SEC-SAXS on FUT8 ( Figure 4B,C ), which conclusively demonstrated that FUT8 exists as a dimer in solution.…”

Glycosyltransferases and non-alcoholic fatty liver disease

“…It plays an important role in the tumorigenesis of non-small cell lung cancer and colon carcinoma [96,97] . Human Fut8 gene is located on chromosome 14q23.3, and consists of at least nine exons spanning more than a 50 kb genomic region, and the coding sequence is divided into eight exons [98,99] .…”