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IQTCUB Researchers reveal the Molecular Mechanism of AaNGT, which is involved in a Significant Protein Modification

The journal Nature Communications has published a study of the molecular mechanism of Aggregatibacter aphrophilus N-glycosyltransferase (AaNGT), an enzyme responsible of a major post-translational modification in proteins of pathogenic bacteria. The multidisciplinary work has been led by a research team in the Institute of Theoretical and Computational Chemistry of the Universitat de Barcelona (IQTCUB) and University of Zaragoza, that combines structural biology, synthetic chemical biology, kinetic experiments and computational chemistry techniques.

The group of Carme Rovira, ICREA Research Professor at the University of Barcelona, together with the group of Ramón Hurtado-Guerrero, ARAID investigator at the Instituto de Biocomputación y Física de Sistemas Complejos (BIFI-Unizar), have solved the 3D structure of the enzyme in complex with several substrates, as well as the mechanism of action of AaNGT, with potential as both a therapeutic target and a biotechnological tool for the synthesis of glycans.

Proteins are biomolecules essential for life. Most proteins need to be altered after being synthesized, a process named post-translational modifications. One of the most important post-translational modification is N-glycosylation, in which a sugar molecule becomes attached to an asparagine residue (amino acid) in the protein. Other sugars can subsequently attach to the initial one, forming complex branches that are crucial for the stability of the protein and its correct functioning. Many N-glycosylated proteins, such as the spike protein of SARS-CoV-2, are therapeutic targets.

N-glycosylation is usually initiated by an oligosaccharyltransferase enzyme (OST), that binds a specific oligosaccharide (a short sugar chain) to specific asparagine residues in proteins. It was recently discovered that a bacterial enzyme, identified as soluble N-glycosyltransferase (NGT), could perform a simpler N-glycosylation using a single sugar molecule, which could have biotechnological applications (e.g. optimization of the glycan decoration of pharmaceuticals).

Although 3D structures of NGTs had been previously reported, as well as mutagenesis experiments, in which amino acids in the active site are replaced to different ones to check their importance to the function of the enzyme, the molecular mechanism remained unknown due to a lack of understanding of enzyme-substrate interactions, and the difficulties in identifying the residues necessary for catalysis. Therefore, the catalytic mechanism employed by the enzyme remained unknown.

The researchers Beatriz Piniello, under the supervision of Carme Rovira from the Department of Inorganic and Organic Chemistry University of Barcelona, Javier Macías-León and Ana García-García, under the supervision of Ramón Hurtado-Guerrero, ARAID investigator at the Instituto de Biocomputación y Física de Sistemas Complejos (BIFI-Unizar) and visitor researcher at the University of Copenhagen, in collaboration with different teams both national and international (Francisco Corzana from University of La Rioja and Atsushi Miyagawa from Nagoya Institute of Technology) used structural biology, biochemical and computational techniques to uncover the 3D structure of AaNGT in complex with several substrates, as well as its molecular mechanism of action.

In the study the researchers discovered that AaNGT uses a particular pair of basic/acidic residues to recognize the amino acid sequence where the crucial asparagine is located. These residues were identified from the 3D structures of AaNGT and an acceptor peptide (a short protein containing the relevant sequence). Furthermore, structures of the enzyme in complex with poor substrates and inhibitors reveal that these molecules bind in non-productive

conformations, explaining their limited catalysis. Finally, the researchers used quantum mechanics/molecular dynamics simulations to uncover the enzyme catalytic mechanism, in which the asparagine residue needs to be in its imidic form to react, and the donor substrate itself, rather than an enzyme residue as other glycosyltransferase enzymes, acts as the general base.

«These findings are not only significant for enhancing our understanding of NGTs but also open up novel mechanistic pathways for achieving glycosylation that diverges from the most established mechaisms in GTs», remarked Beatriz Piniello from the IQTCUB and first author.

«Additionally, the knowledge inferred from this work might serve to engineer these enzymes to use them for biotechnological applications such as the synthesis of customed N-glycans in molecules as important as antibodies», remarked by Hurtado-Guerrero.

Article reference

Beatriz Piniello, Javier Macías-León, Shun Miyazaki, Ana García-García, Ismael Compañón, Mattia Ghirardello, Víctor Taleb, Billy Veloz, Francisco Corzana, Atsushi Miyagawa, Carme Rovira*, and Ramon Hurtado-Guerrero. «Molecular basis for bacterial N-glycosylation by a soluble HMW1C-like N-glycosyltransferase». Nature Communications, 2023. DOI: 10.1038/s41467-023-41238-1 Link: https://rdcu.be/dmq2C