Atomic resolution structure of EhpR: phenazine resistance in Enterobacter agglomerans Eh1087 follows principles of bleomycin/mitomycin C resistance in other bacteria

To understand the resistance mechanism installed by EhpR we have determined its crystal structure in the apo form at 2.15 ？ resolution and in complex with griseoluteic acid at 1.01 ？, respectively. While EhpR shares a common fold with glyoxalase-I/bleomycin resistance proteins, the ligand binding site does not contain residues that some related proteins employ to chemically alter their substrates. Binding of the antibiotic is mediated by π-stacking interactions of the aromatic moiety with the side chains of aromatic amino acids and by a few polar interactions. The dissociation constant KD between EhpR and griseoluteic acid was quantified as 244 ± 45 μM by microscale thermophoresis measurements.The data accumulated here suggest that EhpR confers resistance by binding D-alanyl-griseoluteic acid and acting as a chaperone involved in exporting the antibiotic rather than by altering it chemically. It is tempting to speculate that EhpR acts in concert with EhpJ, a transport protein of the major facilitator superfamily that is also encoded in the phenazine biosynthesis operon of E. agglomerans. The low affinity of EhpR for griseoluteic acid may be required for its physiological function.Newly emerging resistance against antibiotics is an increasing problem in the treatment of infectious disease. The situation is currently worsening at such an alarming speed that the World Health Organization decided to bring it to the spotlight by making it the topic of World Health Day in 2011 [1]. In order to overcome resistance, create opportunities for the development of novel antibiotics or enable the continued use of existing compounds, it is important to understand resistance mechanisms at the molecular level. These mechanisms are highly versatile, from simple mutation of the antibiotic's target to development of mechanisms to reduce uptake by the infectious organism or the installation of factors that destroy or in other ways deactivate the antibiotic [2]. The latter is usually ach