Ligand bound structure of a 6-hydroxynicotinic acid 3-monooxygenase provides mechanistic insights

Zachary R Turlington; Sofia Vaz Ferreira de Macedo; Kay Perry; Sam L Belsky; Jennifer A Faust; Mark J Snider; Katherine A Hicks

doi:10.1016/j.abb.2023.109859

Ligand bound structure of a 6-hydroxynicotinic acid 3-monooxygenase provides mechanistic insights

Arch Biochem Biophys. 2024 Feb:752:109859. doi: 10.1016/j.abb.2023.109859. Epub 2023 Dec 16.

Authors

Zachary R Turlington¹, Sofia Vaz Ferreira de Macedo¹, Kay Perry², Sam L Belsky³, Jennifer A Faust³, Mark J Snider³, Katherine A Hicks⁴

Affiliations

¹ Department of Chemistry, State University of New York at Cortland, Cortland, NY, 13045, United States.
² NE-CAT and Department of Chemistry and Chemical Biology, Cornell University, Argonne National Laboratory, Argonne, IL, United States.
³ Department of Chemistry, The College of Wooster, Wooster, OH, 44691, United States.
⁴ Department of Chemistry, State University of New York at Cortland, Cortland, NY, 13045, United States. Electronic address: katherine.hicks@cortland.edu.

PMID: 38104959
DOI: 10.1016/j.abb.2023.109859

Abstract

6-Hydroxynicotinic acid 3-monooxygenase (NicC) is a bacterial enzyme involved in the degradation of nicotinic acid. This enzyme is a Class A flavin-dependent monooxygenase that catalyzes a unique decarboxylative hydroxylation. The unliganded structure of this enzyme has previously been reported and studied using steady- and transient-state kinetics to support a comprehensive kinetic mechanism. Here we report the crystal structure of the H47Q NicC variant in both a ligand-bound (solved to 2.17 Å resolution) and unliganded (1.51 Å resolution) form. Interestingly, in the liganded form, H47Q NicC is bound to 2-mercaptopyridine (2-MP), a contaminant present in the commercial stock of 6-mercaptopyridine-3-carboxylic acid(6-MNA), a substrate analogue. 2-MP binds weakly to H47Q NicC and is not a substrate for the enzyme. Based on kinetic and thermodynamic characterization, we have fortuitously captured a catalytically inactive H47Q NicC•2-MP complex in our crystal structure. This complex reveals interesting mechanistic details about the reaction catalyzed by 6-hydroxynicotinic acid 3-monooxygenase.

Keywords: Bacterial nicotinic acid metabolism; Flavin adenine dinucleotide (FAD); Flavin monooxygenase; Protein structure.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Flavin-Adenine Dinucleotide* / chemistry
Kinetics
Ligands
Mixed Function Oxygenases* / chemistry

Substances

6-hydroxynicotinate 3-monooxygenase
Ligands
Flavin-Adenine Dinucleotide
Mixed Function Oxygenases