Comparative X-ray analysis of the un-liganded fosfomycin-target MurA

Abstract

MurA, an essential enzyme for the synthesis of the bacterial cell wall, follows an induced-fit mechanism. Upon substrate binding, the active site forms in the interdomain cleft, involving movements of the two domains of the protein and a reorientation of the loop Pro112-Pro121. We compare two structures of un-liganded MurA from Enterobacter cloacae: a new orthorhombic form, solved to 1.80 A resolution, and a monoclinic form, redetermined to 1.55 A resolution. In the monoclinic form, the loop Pro112-Pro121 stretches into solvent, while in the new form it adopts a winded conformation, thereby reducing solvent accessibility of the critical residue Cys115. In the interdomain cleft a network of 27 common water molecules has been identified, which partially shields negative charges in the cleft and stabilizes the orientation of catalytically crucial residues. This could support substrate binding and ease domain movements. Near the hinge region an isoaspartyl residue has been recognized, which is the product of post-translational modification of the genetically encoded Asn67-Gly68. The homogeneous population with L-isoaspartate in both structures suggests that the modification in Enterobacter cloacae MurA is not a mere aging defect but rather the result of a specific in vivo process.

Evidence That the Fosfomycin Target Cys115 in UDP-N-acetylglucosamine Enolpyruvyl Transferase (MurA) Is Essential for Product Release

“…7, b and c). It has been reported previously that the opening of the last turn of helix a2 is a main feature in the transition from the winded conformation of unliganded MurA observed in 1EJC into the U-shaped loop of liganded MurA (30). We propose that transposition of the last turn of the loop-anchoring helix and Asp 123 is the key event for winding up the loop and eventually releasing EP-UNAG.…”

“…The ligands were modeled according to the clear electron density map. Residue 67 of each of the eight molecules was modeled as iso-aspartate (30). The data collection and refinement statistics are summarized in Table I Kinetic Analysis-The catalytic activity of E. cloacae C115S MurA was tested at 20°C with a Shimadzu 1650PC spectrophotometer.…”

Functional Consequence of Covalent Reaction of Phosphoenolpyruvate with UDP-N-acetylglucosamine 1-Carboxyvinyltransferase (MurA)

“…2A, Table 2). This result was unexpected, as we had previously crystallized the same enzyme in distinct open and ligand-free states (22). The only difference between this and our previous studies is that the enzyme was not treated with phosphate buffer before crystallization, as discussed below.…”

Structural and Functional Characterization of NikO, an Enolpyruvyl Transferase Essential in Nikkomycin Biosynthesis

“…Several deviations between the deposited amino acid sequence (UniProt accession code: Q712I1) and the electron density were found upon model building (P52R, D96E, V97L, and V141D). In the homologous enzyme MurA, which had been used as a search template for molecular replacement, residue 67 was found to be an isoaspartic acid (39). This aspartic acid residue is conserved between MurA and NikO (Asp-79), but the electron density clearly showed no isoaspartate formation.…”