Role of K22 and R120 in the covalent binding of the antibiotic fosfomycin and the substrate-induced conformational change in UDP-N-acetylglucosamine enolpyruvyl transferase.
UDP-N-acetylglucosamine enolpyruvyl transferase (MurA), catalyzes the first step in the biosynthesis of peptidoglycan, involving the transfer of the intact enolpyruvyl moiety from phosphoenolpyruvate to the 3'-hydroxyl group of UDP-N-acetylglucosamine (UDPNAG). The enzyme is irreversibly inhibited by the antibiotic fosfomycin. The inactivation is caused by alkylation of a highly conserved cysteine residue (C115) that participates in the binding of phosphoenolpyruvate. The three-dimensional structure of the enzyme suggests that two residues may play a decisive role in fosfomycin binding: K22 and R120. To investigate the role of these residues, we have generated the K22V, K22E, K22R and R120K single mutant proteins as well as the K22V/R120K and K22V/R120V double mutant proteins. We demonstrated that the K22R mutant protein behaves similarly to wild-type enzyme, whereas the K22E mutant protein failed to form the covalent adduct. On the other hand, the K22V mutant protein requires the presence of UDPNAG for the formation of the adduct indicating that UDPNAG plays a crucial role in the organization of productive interactions in the active site. This model receives strong support from heat capacity changes observed for the K22V/R120K and R120K mutant proteins: in both mutant proteins, the heat capacity changes are markedly reduced indicating that their ability to form a closed protein conformation is impeded due to the R120K exchange.
Thomas
Alison M
AM
Eidgenössische Technische Hochschule Zürich, Institute of Plant Sciences, Department of Agricultural and Food Sciences and Department of Biology, Zürich, Switzerland.
Ginj
Cristian
C
Jelesarov
Ilian
I
Amrhein
Nikolaus
N
Macheroux
Peter
P
D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002151:Calorimetry; D017931:DNA Primers; D005578:Fosfomycin / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
D017931:DNA Primers; D005578:Fosfomycin; D019883:Alkyl and Aryl Transferases; C022566:UDP-N-acetylglucosamine 1-carboxyvinyltransferase