Inhibitors of a Na+-pumping NADH-ubiquinone oxidoreductase play multiple roles to block enzyme function

Abstract

The Na+-pumping NADH-ubiquinone (UQ) oxidoreductase (Na+-NQR) is present in the respiratory chain of many pathogenic bacteria and is thought to be a promising antibiotic target. While many details of Na+-NQR structure and function are known, the mechanisms of action of potent inhibitors is not well understood; elucidating the mechanisms would not only advance drug design strategies but might also provide insights on a terminal electron transfer from riboflavin to UQ. To this end, we performed photoaffinity labeling experiments using photoreactive derivatives of two known inhibitors, aurachin and korormicin, on isolated Vibrio cholerae Na+-NQR. The inhibitors labeled the cytoplasmic surface domain of the NqrB subunit including a protruding N-terminal stretch, which may be critical to regulate the UQ reaction in the adjacent NqrA subunit. The labeling was blocked by short-chain UQs such as ubiquinone-2. The photolabile group (2-aryl-5-carboxytetrazole (ACT)) of these inhibitors reacts with nucleophilic amino acids, so we tested mutations of nucleophilic residues in the labeled region of NqrB, such as Asp49 and Asp52 (to Ala), and observed moderate decreases in labeling yields, suggesting that these residues are involved in the interaction with ACT. We conclude that the inhibitors interfere with the UQ reaction in two ways: the first is blocking structural rearrangements at the cytoplasmic interface between NqrA and NqrB, and the second is the direct obstruction of UQ binding at this interfacial area. Unusual competitive behavior between the photoreactive inhibitors and various competitors corroborates our previous proposition that there may be two inhibitor binding sites in Na+-NQR.

Cryo-EM structures of Na+-pumping NADH-ubiquinone oxidoreductase from Vibrio cholerae

“…This is probably because the binding site of UQ in NqrA, which may be located near the inhibitor binding site (18), is influenced by the mutation. While IC50 values of korormicin A and aurachin D-42 determined in the NADH-UQ1 oxidoreductase assay with the wild-type enzyme were one-digit nM levels (18,19), 50% inhibition was not observed for either inhibitor with the mutant, even at 2.0 µM.…”

“…Based on photoaffinity labeling experiments (18,19), we previously showed that korormicin A (a highly specific inhibitor of Na + -NQR, 18,20) and aurachin D-type inhibitors (naphthoquinone-like compounds) bind to a part of the protruding N-terminal stretch starting with transmembrane helix (TMH) 1 of NqrB (Try23−Lys54, Supplementary Figure 1) and that the UQ head-ring binds to the cytoplasmic region of NqrA close to the N-terminal stretch of NqrB (Leu32−Met39 and Phe131−Lys138, Supplementary Figure 1). Thus, the N-terminal stretch of NqrB (Met1−Lys54) may be critical for regulating the UQ reaction at the adjacent NqrA.…”

Molecular dynamics modeling of the Vibrio cholera Na+-translocating NADH:quinone oxidoreductase NqrB–NqrD subunit interface

“…Moreover, the interface between NqrB and NqrA is itself of considerable interest. Masuya et al [ 44 ] used photoaffinity labeling studies to document inhibitor binding near a ubiquinone binding site [ 45 ] at this interface, proposing that the residues identified by Tuz et al [ 16 ] influence ubiquinone binding via indirect effects that lead to long-range structural changes.…”

Inhibition of bacterial FMN transferase: A potential avenue for countering antimicrobial resistance

“…1,[13][14][15] Disruption of the chemiosmotic gradient generated by these redox complexes has the potential to arrest essential bacterial functions such as ATP synthesis, flagellar rotation, and nutrient transport. 11,16,17 Indeed, interruption of the Ftp gene in Listeria monocytogenes limited the bacterium's ability to grow on a carbon source that requires extracellular electron transport for utilization. 18 Thus, inhibitors of FMNylation (i.e., of Ftp) have the potential to curtail bacterial growth and therefore could lead to new antimicrobials to combat antibiotic-resistant bacteria, which are a growing menace.…”

“…It is now appreciated that Ftp's FMN‐transferase activity is required for the periplasmic FMNylation and electron‐transfer functions of subunits (e.g., NqrC and RnfG) of these flavin‐based cytoplasmic membrane‐bound NQR and/or RNF redox complexes, providing a link between those periplasmic functions and the redox reactions that occur within the bacterial cytoplasm 1,13–15 . Disruption of the chemiosmotic gradient generated by these redox complexes has the potential to arrest essential bacterial functions such as ATP synthesis, flagellar rotation, and nutrient transport 11,16,17 . Indeed, interruption of the Ftp gene in Listeria monocytogenes limited the bacterium's ability to grow on a carbon source that requires extracellular electron transport for utilization 18 .…”