Atomic resolution structure of EhpR

TY - JOUR

T1 - Atomic resolution structure of EhpR

T2 - phenazine resistance in Enterobacter agglomerans Eh1087 follows principles of bleomycin/mitomycin C resistance in other bacteria

AU - Yu, Shen

AU - Vit, Allegra

AU - Devenish, Sean

AU - Mahanty, H Khris

AU - Itzen, Aymelt

AU - Goody, Roger S

AU - Blankenfeldt, Wulf

PY - 2011/8/17

Y1 - 2011/8/17

N2 - BACKGROUND: The phenazines are redox-active secondary metabolites that a large number of bacterial strains produce and excrete into the environment. They possess antibiotic activity owing to the fact that they can reduce molecular oxygen to toxic reactive oxygen species. In order to take advantage of this activity, phenazine producers need to protect themselves against phenazine toxicity. Whereas it is believed that phenazine-producing pseudomonads possess highly active superoxide dismutases and catalases, it has recently been found that the plant-colonizing bacterium Enterobacter agglomerans expresses a small gene ehpR to render itself resistant towards D-alanyl-griseoluteic acid, the phenazine antibiotic produced by this strain.RESULTS: To understand the resistance mechanism installed by EhpR we have determined its crystal structure in the apo form at 2.15 Å resolution and in complex with griseoluteic acid at 1.01 Å, 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.CONCLUSIONS: 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.

AB - BACKGROUND: The phenazines are redox-active secondary metabolites that a large number of bacterial strains produce and excrete into the environment. They possess antibiotic activity owing to the fact that they can reduce molecular oxygen to toxic reactive oxygen species. In order to take advantage of this activity, phenazine producers need to protect themselves against phenazine toxicity. Whereas it is believed that phenazine-producing pseudomonads possess highly active superoxide dismutases and catalases, it has recently been found that the plant-colonizing bacterium Enterobacter agglomerans expresses a small gene ehpR to render itself resistant towards D-alanyl-griseoluteic acid, the phenazine antibiotic produced by this strain.RESULTS: To understand the resistance mechanism installed by EhpR we have determined its crystal structure in the apo form at 2.15 Å resolution and in complex with griseoluteic acid at 1.01 Å, 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.CONCLUSIONS: 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.

KW - Anti-Bacterial Agents

KW - Bacteria

KW - Bacterial Proteins

KW - Bleomycin

KW - Crystallography, X-Ray

KW - Databases, Protein

KW - Dimerization

KW - Drug Resistance, Bacterial

KW - Enterobacter

KW - Lactoylglutathione Lyase

KW - Mitomycin

KW - Phenazines

KW - Protein Binding

KW - Protein Structure, Tertiary

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1186/1472-6807-11-33

DO - 10.1186/1472-6807-11-33

M3 - SCORING: Journal article

C2 - 21849072

VL - 11

SP - 33

JO - BMC STRUCT BIOL

JF - BMC STRUCT BIOL

SN - 1472-6807

ER -