Fusarium verticillioides NAT1 (FDB2) N-malonyltransferase is structurally, functionally, and phylogenetically distinct from its N-acetyltransferase (NAT) homologues

Authors: KARAGIANNI, ELENI-PAVLINA - Democritus University Of Thrace; KONTOMINA, EVANTHIA - Democritus University Of Thrace; LOWE, EDWARD - University Of Oxford; ATHANASOPOULOS, KONSTANTINOS - Democritus University Of Thrace; PAPANIKOLAOU, GEORGIA - Democritus University Of Thrace; GAREFALAKI, VASILIKI - Democritus University Of Thrace; KOTSELI, VARVARA - Democritus University Of Thrace; ZALIOU, SOFIA - Democritus University Of Thrace; GRIMAUD, TOM - Democritus University Of Thrace; ARVANITI, KONSTANTINA - Democritus University Of Thrace; TSATIRI, MARIA-AGGELIKI - Democritus University Of Thrace; FAKIS, GIANNOULIS - Democritus University Of Thrace; Glenn, Anthony - Tony; ROVERSI, PIETRO - University Of Leicester; ABUHAMMAD, AREEJ - University Of Oxford; RYAN, ALI - University Of Oxford; SIM, ROBERT - University Of Oxford; SIM, EDITH - University Of Oxford; BOUKOUVALA, SOTIRIA - Democritus University Of Thrace

Submitted to: FEBS Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/29/2022
Publication Date: 10/18/2022
Citation: Karagianni, E., Kontomina, E., Lowe, E., Athanasopoulos, K., Papanikolaou, G., Garefalaki, V., Kotseli, V., Zaliou, S., Grimaud, T., Arvaniti, K., Tsatiri, M., Fakis, G., Glenn, A.E., Roversi, P., Abuhammad, A., Ryan, A., Sim, R., Sim, E., Boukouvala, S. 2022. Fusarium verticillioides NAT1 (FDB2) N-malonyltransferase is structurally, functionally, and phylogenetically distinct from its N-acetyltransferase (NAT) homologues. FEBS Journal. 290:2412-2436. https://doi.org/10.1111/febs.16642.
DOI: https://doi.org/10.1111/febs.16642

Interpretive Summary: Species of Fusarium are well known fungal plant pathogens causing extensive damage to many agriculturally important crops. Further, many of the species can produce toxins that cause a variety of illnesses in animals and humans. For example, Fusarium verticillioides is often the most significant fungal pathogen of corn. It causes a moldy rot on the kernels and contaminates them with toxins. This fungus also has evolved the ability to resist and detoxify antimicrobial compounds produced by corn, and a key enzyme used by F. verticillioides is NAT1, which is an N-malonyltransferase. This NAT1 enzyme was studied in detail using various techniques, and the structure of the enzyme was determined. NAT1 has a distinctive structure different from most known NAT enzymes, including two other NAT enzymes produced by F. verticillioides noted as NAT2 and NAT3. Given the role of NAT1 in detoxifying the corn antimicrobial compounds, this study provides greater understanding of the biochemistry and biological significance of NAT1 and may result in more targeted strategies to control the fungus.

Technical Abstract: Fusarium endophytes damage cereal crops and contaminate produce with mycotoxins. Those fungi overcome the main chemical defence of host via detoxification by a malonyl-CoA dependent enzyme homologous to xenobiotic metabolizing arylamine N-acetyltransferase (NAT). In Fusarium verticillioides (teleomorph Gibberella moniliformis, GIBMO), this N-malonyltransferase activity is attributed to (GIBMO)NAT1, and the fungus has two additional isoenzymes, (GIBMO)NAT3 (N-acetyltransferase) and (GIBMO)NAT2 (unknown function). We present the crystallographic structure of (GIBMO)NAT1, also modeling other fungal NAT homologues. Monomeric (GIBMO)NAT1 is distinctive, with access to the catalytic core through two "tunnel-like" entries separated by a "bridge-like" helix. In the quaternary arrangement, (GIBMO)NAT1 monomers interact in pairs along an extensive interface whereby one entry of each monomer is covered by the N-terminus of the other monomer. Although monomeric (GIBMO)NAT1 apparently accommodates acetyl-CoA better than malonyl-CoA, dimerization changes the active site to allow malonyl-CoA to reach the catalytic triad (Cys110, His158, Asp173) via the single uncovered entry, and anchor its terminal carboxyl-group via hydrogen bonds to Arg109, Asn157 and Thr261. Lacking a terminal carboxyl-group, acetyl-CoA cannot form such stabilizing interactions, while longer acyl-CoAs enter the active site but cannot reach catalytic Cys. Other NAT isoenzymes lack such structural features, with (GIBMO)NAT3 resembling bacterial NATs and (GIBMO)NAT2 adopting a structure intermediate between (GIBMO)NAT1 and (GIBMO)NAT3. Biochemical assays confirmed differential donor substrate selectivity of (GIBMO)NAT isoenzymes, with phylogenetic analysis demonstrating evolutionary separation. Given the role of (GIBMO)NAT1 in enhancing Fusarium pathogenicity, unravelling the structure and function of this enzyme may benefit research into more targeted strategies for pathogen control.