Database accession: MF7000505
Name: Klebsiella pneumoniae fosfomycin-resistance protein (FosAKP)
PDB ID: 8r37
Experimental method: X-ray (1.48 Å)
Assembly: Homodimer
Source organism: Klebsiella pneumoniae
Primary publication of the structure:
Varotsou C, Ataya F, Papageorgiou AC, Labrou NE
Structural Studies of Fosfomycin-Resistance Protein and Its Application for the Development of an Optical Biosensor for Fosfomycin Determination.
(2023) Int J Mol Sci 25:
PMID: 38203259
Abstract:
Fosfomycin-resistance proteins (FosAs) are dimeric metal-dependent glutathione transferases that conjugate the antibiotic fosfomycin (Fos) to the tripeptide glutathione (γ-Glu-Cys-Gly, GSH), rendering it inactive. In the present study, we reported a comparative analysis of the functional features of two FosAs from Pseudomonas aeruginosa (FosAPA) and Klebsiella pneumoniae (FosAKP). The coding sequences of the enzymes were cloned into a T7 expression vector, and soluble active enzymes were expressed in E. coli. FosAKP displayed higher activity and was selected for further studies. The crystal structure of the dimeric FosAKP was determined via X-ray crystallography at 1.48 Å resolution. Fos and tartrate (Tar) were found bound in the active site of the first and second molecules of the dimer, respectively. The binding of Tar to the active site caused slight rearrangements in the structure and dynamics of the enzyme, acting as a weak inhibitor of Fos binding. Differential scanning fluorimetry (DSF) was used to measure the thermal stability of FosAKP under different conditions, allowing for the selection of a suitable buffer to maximize enzyme operational stability. FosAKP displays absolute specificity towards Fos; therefore, this enzyme was exploited for the development of an enzyme-based colorimetric biosensor. FosAKP was tethered at the bottom of a plastic cuvette using glutaraldehyde chemistry to develop a simple colorimetric method for the determination of Fos in drinking water and animal plasma.
Annotations from the GeneOntology database. Only terms that fit at least two of the interacting proteins are shown. Molecular function:
glutathione transferase activity glutathione transferase activity
metal ion binding metal ion binding
Biological process: not assigned
Cellular component: not assigned
Structural annotations of the participating protein chains.Entry contents: 2 distinct polypeptide molecules
Chains: A, B
Notes: All chains according to the most probable oligomerization state stored in PDBe were considered.
Number of unique protein segments: 1
Name: FosA family fosfomycin resistance glutathione transferase
Source organism: Klebsiella pneumoniae
Length: 139 residues
Sequence:Sequence according to the corresponding UniProt protein segmentMLSGLNHLTLAVSQLAPSVAFYQQLLGMTLHARWDSGAYLSCGDLWLCLSLDPQRRVTPPEESDYTHYAFSISEADFASFAARLEAAGVAVWKLNRSEGASHYFLDPDGHKLELHVGSLAQRLAACREQPYKGMVFFEQ
UniProtKB AC: A0A086IRG1 (positions: 1-139)
Coverage: 100%
Name: FosA family fosfomycin resistance glutathione transferase
Source organism: Klebsiella pneumoniae
Length: 139 residues
Sequence:Sequence according to the corresponding UniProt protein segmentMLSGLNHLTLAVSQLAPSVAFYQQLLGMTLHARWDSGAYLSCGDLWLCLSLDPQRRVTPPEESDYTHYAFSISEADFASFAARLEAAGVAVWKLNRSEGASHYFLDPDGHKLELHVGSLAQRLAACREQPYKGMVFFEQ
UniProtKB AC: A0A086IRG1 (positions: 1-139)
Coverage: 100%
Evidence demonstrating that the participating proteins are unstructured prior to the interaction and their folding is coupled to binding. Representative domain in related structures: Glyoxalase/Bleomycin resistance protein/Dioxygenase superfamily
Evidence level: Indirect evidence
Evidence coverage: The full structure participates in mutual synergistic folding.
Complex Evidence:
The VOC superfamily of metalloenzymes is characterized by a three-dimensional domain-swapped arrangement of tandem βαβββ-motifs (PMID:24447055). The original gene duplication event led to the βαβββ tandem structure, which appears to require dimerization for stability. Two different forms of domain-swapped dimers may coexist in solution (PMID:12121648) in which both subunits of the homodimer participate in coordination of each metal ion and formation of the U-shaped active sites in the enzyme (PMID:24004181). The complex is predominantly dimeric in solution (gel filtration) (PMID:12121648).
Chain B:
N/A
Chain A:
N/A
Surface and contacts features:
Structures from the PDB that contain the same number of proteins, and the proteins from the two structures show a sufficient degree of pairwise similarity, i.e. they belong to the same UniRef90 cluster (the full proteins exhibit at least 90% sequence identity) and convey roughly the same region to their respective interactions (the two regions from the two proteins share a minimum of 70% overlap). Download the CIF file (.cif)
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