Substrate Engagement and Catalytic Mechanisms of N-Acetylglucosaminyltransferase V

Abstract

α-Mannoside β-1,6-N-acetylglucos­aminyl­transferase V (MGAT5) is a mammalian glycosyltransferase involved in complex N-glycan formation, which strongly drives cancer when overexpressed. Despite intense interest, the catalytic mechanism of MGAT5 is not known in detail, precluding therapeutic exploitation. We solved structures of MGAT5 complexed to glycosyl donor and acceptor ligands, revealing an unforeseen role for donor-induced loop rearrangements in controlling acceptor substrate engagement. QM/MM metadynamics simulations of MGAT5 catalysis highlight the key assisting role of Glu297 and reveal considerable conformational distortions imposed upon the glycosyl donor during transfer. Detailed mechanistic characterization of MGAT5 will aid inhibitor development to correct cancer-associated N-glycosylation.

N-acetylglucosaminyltransferase-V requires a specific noncatalytic luminal domain for its activity toward glycoprotein substrates

“…This is consistent with previous study in which GnT-V mutants lacking a part of the N domain fully retained their activity toward oligosaccharides ( 27 ). In addition, a soluble GnT-V catalytic domain that lacks the whole N domain and was used for structural studies is still active toward oligosaccharides ( 20 , 21 ). These data demonstrated that the N domain is not involved in a monosaccharide transfer reaction itself.…”

“…The other possibility is that the N domain could change the conformation of the catalytic domain by moving like a pendulum. As the N domain is connected to the neighboring domain via two highly flexible loops ( 19 , 21 ), movement of the N domain might alter the conformation of the catalytic region for efficient product release. However, if this putative conformational change induced by the N domain is a prerequisite for GnT-V action, GnT-VΔN would lose activity regardless of the type of substrate.…”

“…Similar to other glycosyltransferases, GnT-V has a type II membrane topology composed of a short N-terminal cytosolic region, a transmembrane domain, a stem region, and a large C-terminal catalytic region ( 19 ). The crystal structures of the luminal region of GnT-V solved by us and another group allowed the elucidation of the catalytic residue and the binding sites for the acceptor and donor substrates ( 20 , 21 ). In addition, our recent study using a structural model of a GnT-V-substrate complex and a series of point mutants suggested that a few amino acid residues outside the catalytic pocket are involved in recognizing the glycan core or polypeptide part of the acceptor substrate ( 22 ).…”

Glycosylation: Rising Potential for Prostate Cancer Evaluation

“…The raised level of GlcNAc-branching N-glycans is a result of increased activity of mannoside N-acetylglucosaminyltransferase 5 (GnT-V), which is encoded by the MGAT5 gene, often activated in cancer cells [59]. Activity of GnT-V leads to the formation of complexed tri-and tetra-antennary structures (Figure 1A), which can affect the stability, functional activity and half-life of proteins, as well as membrane dynamics [61,103]. Furthermore, branched N-glycans can be modified via β1,4-galactosyltransferases (β1,4-GalTs), thus elongated with poly-N-acetyllactosamine repeats, and finally capped with sialic acid or fucose.…”

Challenges and limitations in the studies of glycoproteins: A computational chemist's perspective

“…Another interesting study was recently reported by Darby et al, who analyzed the α‐mannoside β‐1,6‐ N ‐acetylglucosaminyltransferase V (MGAT5), which is a mammalian glycosyltransferase contributing to N ‐glycan formation. MGAT5 is known to strongly drive cancer when overexpressed 101 . The catalytic mechanism of MGAT5 was unknown, precluding therapeutic exploitation.…”