Mutation of a distal gating residue modulates NADH binding in NADH:Quinone oxidoreductase from Pseudomonas aeruginosa PAO1.

Enzymes require flexible regions to adopt multiple conformations during catalysis. The mobile regions of enzymes include gates that modulate the passage of molecules in and out of the enzyme's active site. The enzyme PA1024 from Pseudomonas aeruginosa PA01 is a recently discovered flavin-dependent NADH:quinone oxidoreductase (NQO, EC 1.6.5.9). Q80 in loop 3 (residues 75-86) of NQO is ∼15 Å away from the flavin and creates a gate that seals the active site through a hydrogen bond with Y261 upon NADH binding. In this study, we mutated Q80 to glycine, leucine, or glutamate to investigate the mechanistic significance of distal residue Q80 in NADH binding in the active site of NQO. The UV-visible absorption spectrum reveals that the mutation of Q80 minimally affects the protein microenvironment surrounding the flavin. The anaerobic reductive half-reaction of the NQO-mutants yields a ≥25-fold increase in the Kd value for NADH compared to the WT enzyme. However, we determined that the kred value was similar in the Q80G, Q80L, and wildtype enzymes and only ∼25% smaller in the Q80E enzyme. Steady-state kinetics with NQO-mutants and NQO-WT at varying concentrations of NADH and 1,4-benzoquinone establish a ≤5-fold decrease in the kcat/KNADH value. Moreover, there is no significant difference in the kcat/KBQ (∼1 × 106 M-1s-1) and kcat (∼24 s-1) values in NQO-mutants and NQO-WT. These results are consistent with the distal residue Q80 being mechanistically essential for NADH binding to NQO with minimal effect on the quinone binding to the enzyme and hydride transfer from NADH to flavin.

Identifying continuous pores in protein structures with PROPORES by computational repositioning of gating residues.

Proteins containing concavities such as pockets, cavities, and tunnels or pores perform important functions in ligand-induced signal transduction, enzymatic catalysis, and in facilitating the permeation of small molecules through membranes. Computational algorithms for identifying such shapes are therefore of great use for studying the mechanisms of these reactions. We developed the novel toolkit PROPORES for pore identification and applied our program to the systems aquaporin, tryptophan synthase, leucine transporter, and acetylcholinesterase. As a novel feature, the program checks whether access to occluded ligand binding pockets or blocked channels can be achieved by systematically rotating side chains of the gating residues. In this way, we obtain a more flexible view of the putative structural adaptability of protein structures. For the four systems mentioned, the new method was able to identify connections between pores that are separated in the X-ray structures or to connect internal pores with the protein surrounding. The software is available from http://gepard.bioinformatik.uni-saarland.de/software/propores/.