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

• Published in: Archives of biochemistry and biophysics 2024-02, Vol.752 (C), p.109859-109859, Article 109859
• Main Authors: Turlington, Zachary R, Vaz Ferreira de Macedo, Sofia, Perry, Kay, Belsky, Sam L, Faust, Jennifer A, Snider, Mark J, Hicks, Katherine A
• Format: Article
• Language: English
• Subjects: Flavin-Adenine Dinucleotide - chemistry; Kinetics; Ligands; Mixed Function Oxygenases - chemistry
• ISSN: 0003-9861; 1096-0384; 1096-0384
• DOI: 10.1016/j.abb.2023.109859

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.