PMC

Chemical mechanism of β-LS based on structural and kinetic data.

Schematic diagram of the proposed oxyanion intermediate or transition state modeled into the β-LS active site.

Mechanistically important hydrogen bonding interactions in the CMA-AMP/PP_i/Mg²⁺ active site. The conformations of Tyr-326, Tyr-348, and Lys-443 differ from those observed in the apo structure (superimposed in green).

Reactions catalyzed by β-LS and AS-B. β-LS cyclizes CEA to DGPC. Reactions catalyzed by other enzymes result in the formation of clavulanic acid. The truncated substrate analog CMA is a dead-end inhibitor. AS-B converts glutamine and aspartic acid to glutamic acid and asparagine.

Stereo views of ligands bound in the β-LS active site. Only amino acid residues directly coordinated to the Mg²⁺ ions are shown. Solvent molecules are shown as red spheres. (A) ATP/Mg²⁺ structure. (B) Previously determined CEA⋅AMP-CPP/Mg²⁺ structure. (C) CMA-AMP/PP_i/Mg²⁺ structure. (D) DGPC⋅AMP/PP_i/Mg²⁺ structure.

Electron density maps showing bound ligands in the three soaked crystals. The Mg²⁺ ions are shown as gray spheres. (A) ATP/Mg²⁺ structure. Stereo view of a simulated annealing omit map contoured at 2σ with the ATP and Mg²⁺ ions omitted. (B) CMA-AMP/PP_i/Mg²⁺ structure. Stereo view of a simulated annealing omit map contoured at 2σ with the CMA-adenylate, PP_i, and Mg²⁺ ions omitted. (C) DGPC⋅AMP/PP_i/Mg²⁺ structure. Stereo view of a simulated annealing omit map contoured at 2σ with the DGPC, AMP, and PP_i omitted.