Substrate and Inhibitor-Induced Conformational Changes in the Structurally Related Enzymes UDP-N-Acetylglucosamine Enolpyruvyl Transferase (MurA) and 5-Enolpyruvylshikimate 3-Phosphate Synthase (EPSPS)

Abstract

UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) and 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) have both a unique three-dimensional topology and overall reaction mechanism in common. In the case of MurA, the substrate-free, unliganded protein exhibits an "open" conformation. Upon binding of substrates, the protein forms a much more tightly packed so-called "closed" form following an induced fit mechanism. In this closed form, the substrates are properly positioned for catalysis. On the basis of the structural and mechanistic similarities of MurA and EPSPS, a similar conformational change is likely to occur in EPSPS to generate a catalytically competent active site. However, there is currently little experimental evidence available to support the occurrence of such a conformational change in EPSPS. Using limited tryptic digestion of MurA,(1) it could be shown that formation of the "closed" conformation of MurA is accompanied by a marked increase of stability toward proteolytic degradation. Formation of the closed conformation was achieved by addition of either an excess of both substrates or the sugar nucleotide substrate in conjunction with the antibiotic fosfomycin. Analysis of the MurA tryptic fragments by MALDI-TOF mass spectrometry demonstrates that the protection of the protein in either case is caused by (1) a specific shielding of regions thereby becoming less accessible as a result of the conformational change, and (2) an unspecific overall protection of the whole protein due to an apparently reduced flexibility of the peptide backbone in the binary and ternary complexes. The establishment of methods to describe the effects of tryptic digestion on MurA under various conditions was then extended to EPSPS. Although EPSPS was found to be much more stable toward proteolysis than MurA, the presence of shikimate 3-phosphate (S3P) and the inhibitor glyphosate led to a pronounced suppression of proteolytic degradation. When unliganded EPSPS was treated with trypsin, three of the peptide fragments obtained could be identified by mass spectrometry. Two of these are located in a region corresponding to the "catalytic" loop in MurA which participates in the conformational change. This indicates a conformational change in EPSPS, similar to the one observed in MurA, leading to the protection mentioned above. Corroborating evidence was obtained using a conformational sensitive monoclonal antibody against EPSPS which showed a 20-fold reduced affinity toward the protein complexed with S3P and glyphosate as compared to the unliganded enzyme.

Recruitment of genes and enzymes conferring resistance to the nonnatural toxin bromoacetate

“…1B) in the presence of a saturating amount of UDP-GlcNAc (1 mM) in 100 mM triethanolamine-HCl at pH 8.0. Binding of UDP-GlcNAc to MurA has been shown to induce a conformational change resulting in an increased affinity toward PEP (25). The inactivation rate of MurA with fosfomycin is also dependent upon UDP-GlcNAc (8).…”

Chemical shift mapping of shikimate‐3‐phosphate binding to the isolated N‐terminal domain of 5‐enolpyruvylshikimate‐3‐phosphate synthase

“…A partial conformational change upon S3P binding is supported by the results of Stepanek [16], who found that monoclonal antibodies could be raised against the binary EPSPS b S3P complex, distinct from antibodies that recognized the enzyme alone. Also, Krekel et al [17] found that S3P binding did not increase the dissociation constant of a monoclonal antibody raised against EPSP synthase, implying that S3P alone did not induce a full conformational change that disrupted the recognition of the antibody. This contrasted with the results of their mass spectrometric analysis, and could be explained by hypothesizing that S3P induced only a partial conformational change that allowed the antibody access to its epitope while restricting the access of trypsin to proteolytic cleavage sites.…”

Closing down on glyphosate inhibition—with a new structure for drug discovery

“…On ligand binding, the MurA tertiary structure changes from the ''open'' form of the enzyme to the ''closed'' form, in which the two domains rearrange substantially into a tightly packed conformation (13). Using limited tryptic digestion and matrix-assisted laser desorption ionizationtime-of-flight mass spectroscopy, Krekel et al (14) have suggested that binding of S3P in combination with glyphosate also stabilizes EPSPS in a closed conformation. In addition, fluorescence spectroscopy studies imply that the association of glyphosate with the EPSPS-S3P binary complex induces a substantial conformational change in the enzyme (15).…”