Steric hindrance controls pyridine nucleotide specificity of a flavin-dependent NADH:quinone oxidoreductase

Abstract

The crystal structure of the NADH:quinone oxidoreductase PA1024 has been solved in complex with NAD⁺ to 2.2 Å resolution. The nicotinamide C4 is 3.6 Å from the FMN N5 atom, with a suitable orientation for facile hydride transfer. NAD⁺ binds in a folded conformation at the interface of the TIM-barrel domain and the extended domain of the enzyme. Comparison of the enzyme-NAD⁺ structure with that of the ligand-free enzyme revealed a different conformation of a short loop (75-86) that is part of the NAD⁺ -binding pocket. P78, P82, and P84 provide internal rigidity to the loop, whereas Q80 serves as an active site latch that secures the NAD⁺ within the binding pocket. An interrupted helix consisting of two α-helices connected by a small three-residue loop binds the pyrophosphate moiety of NAD⁺ . The adenine moiety of NAD⁺ appears to π-π stack with Y261. Steric constraints between the adenosine ribose of NAD⁺ , P78, and Q80, control the strict specificity of the enzyme for NADH. Charged residues do not play a role in the specificity of PA1024 for the NADH substrate.

Keywords: FMN; NAD+; NADH:quinone oxidoreductase; PA1024; coenzyme; flavoprotein; hydride transfer; pyridine nucleotide; steric hindrance.