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.
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