Donor substrate binding and enzymatic mechanism of human core α1,6-fucosyltransferase (FUT8)

• Published in: Biochimica et biophysica acta Vol. 1820; no. 12; pp. 1915 - 1925
• Main Authors: Kötzler, Miriam P., Blank, Simon, Bantleon, Frank I., Spillner, Edzard, Meyer, Bernd
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.12.2012
• Subjects: biosynthesis; carcinogenesis; Catalysis; colorectal neoplasms; enzyme activity; epitopes; fucose; Fucosyltransferase; Fucosyltransferases - chemistry; Fucosyltransferases - genetics; Fucosyltransferases - isolation & purification; Fucosyltransferases - metabolism; GDP-fucose; Glycosyltransferase; guanine; Guanosine Diphosphate Fucose - metabolism; hepatoma; host-pathogen relationships; Humans; hydrogen bonding; inflammation; Magnetic Resonance Spectroscopy; Models, Molecular; molecular dynamics; Molecular Dynamics Simulation; nuclear magnetic resonance spectroscopy; polysaccharides; prognosis; Protein Conformation; Saturation transfer difference nuclear magnetic resonance; Substrate recognition; Surface Plasmon Resonance; STD; GT-B; Glycosyltransferase; SPC; Saturation transfer difference nuclear magnetic resonance; ADP; OPLS; BSA; FUT8; IDP; SPR; MD; GDP-fucose; GlcNAc; T1; FID; EGF; XDP; MS; RMSF; PME; cePOFUT1; RMSD; Fucosyltransferase; Molecular dynamics simulation; HEK; MALDI TOF; MWCO; NMR; NodZ; VGF; Fcγ; TGFβ; GDP-Fuc; Mes; Substrate recognition
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2012.08.018

Fucosylation is essential for various biological processes including tumorigenesis, inflammation, cell–cell recognition and host–pathogen interactions. Biosynthesis of fucosylated glycans is accomplished by fucosyltransferases. The enzymatic product of core α1,6-fucosyltransferase (FUT8) plays a major role in a plethora of pathological conditions, e.g. in prognosis of hepatocellular carcinoma and in colon cancer. Detailed knowledge of the binding mode of its substrates is required for the design of molecules that can modulate the activity of the enzyme. We provide a detailed description of binding interactions of human FUT8 with its natural donor substrate GDP-fucose and related compounds. GDP-Fuc was placed in FUT8 by structural analogy to the structure of protein-O-fucosyltransferase (cePOFUT) co-crystallized with GDP-Fuc. The epitope of the donor substrate bound to FUT8 was determined by STD NMR. The in silico model is further supported by experimental data from SPR binding assays. The complex was optimized by molecular dynamics simulations. Guanine is specifically recognized by His363 and Asp453. Furthermore, the pyrophosphate is tightly bound via numerous hydrogen bonds and contributes affinity to a major part. Arg365 was found to bind both the β-phosphate and the fucose moiety at the same time. Discovery of a novel structural analogy between cePOFUT and FUT8 allows the placement of the donor substrate GDP-Fuc. The positioning was confirmed by various experimental and computational techniques. The model illustrates details of the molecular basis of substrate recognition for a human fucosyltransferase for the first time and, thus, provides a basis for structure-based design of inhibitors. ► First detailed model of donor substrate binding for human fucosyltransferase FUT8. ► Model is based on X-ray structures combined with ligand-based NMR data and SPR data. ► Refinement with molecular dynamics simulation yields substrate binding mode. ► Major donor binding of FUT8 originates from β-phosphate group. ► The key catalytic residue Arg365 orientates fucose residue and assists GDP release.