Generalized enzymatic mechanism of catalysis by tetrameric l-asparaginases from mesophilic bacteria

The mechanism of catalysis by the l-glutaminase-asparaginase from Pseudomonas 7A (PGA) was investigated using structural, mass spectrometry, and kinetic data. We had previously proposed mechanism of hydrolysis of l-Asn by the type II l-asparaginase from E. coli (EcAII), but that work was limited to just one enzyme. Based on results presented in this report, we postulate that all homotetrameric l-asparaginases from mesophilic bacteria utilize a common ping-pong mechanism of catalysis consisting of two subsequent nucleophilic substitutions. Several new structures of non-covalent complexes of PGA with different substrates, as well as structures of covalent acyl-enzyme intermediates of PGA with canonical substrates (l-Asp and l-Glu) and an opportunistic ligand, a citrate anion, were determined. The results of kinetic experiments monitored by high-resolution LC/MS, when combined with new structural data, clearly show that the reaction catalyzed by l-glutaminase-asparaginases proceeds through formation of a covalent intermediate, as observed previously for EcAII. Additionally, by showing that the same mechanism applies to l-Asn and l-Gln, we postulate that it is common for all these structurally related enzymes.

Structural Insights into the Mechanism of Carbapenemase Activity of the OXA-48 β-Lactamase

ABSTRACT Carbapenem-hydrolyzing class D carbapenemases (CHDLs) are enzymes that produce resistance to the last-resort carbapenem antibiotics, severely compromising the available therapeutic options for the treatment of life-threatening infections. A broad variety of CHDLs, including OXA-23, OXA-24/40, and OXA-58, circulate in Acinetobacter baumannii, while the OXA-48 CHDL is predominant in Enterobacteriaceae. Extensive structural studies of A. baumannii enzymes have provided important information regarding their interactions with carbapenems and significantly contributed to the understanding of the mechanism of their carbapenemase activity. However, the interactions between carbapenems and OXA-48 have not yet been elucidated. We determined the X-ray crystal structures of the acyl-enzyme complexes of OXA-48 with four carbapenems, imipenem, meropenem, ertapenem, and doripenem, and compared them with those of known carbapenem complexes of A. baumannii CHDLs. In the A. baumannii enzymes, acylation by carbapenems triggers significant displacement of one of two conserved hydrophobic surface residues, resulting in the formation of a channel for entry of the deacylating water into the active site. We show that such a channel preexists in apo–OXA-48 and that only minor displacement of the conserved hydrophobic surface residues occurs upon the formation of OXA-48 acyl-enzyme intermediates. We also demonstrate that the extensive hydrophobic interactions that occur between a conserved hydrophobic bridge of the A. baumannii CHDLs and the carbapenem tails are lost in OXA-48 in the absence of an equivalent bridge structure. These data highlight significant differences between the interactions of carbapenems with OXA-48 and those with A. baumannii enzymes and provide important insights into the mechanism of carbapenemase activity of the major Enterobacteriaceae CHDL, OXA-48.

Identification of Nucleophilic Probes for Protease-Mediated Transpeptidation

Proteases have evolved to mediate the hydrolysis of peptide bonds but may perform transpeptidation in the presence of a proper nucleophilic molecule that can effectively compete with water to react with the acyl-enzyme intermediate. There have been several examples of protease-mediated transpeptidation, but they are generally inefficient, and little effort has been made to systematically control the transpeptidation activity of other proteases with good nucleophiles. Here, we developed an on-bead screening approach to find a probe that functions efficiently as a nucleophile in the protease-mediated transpeptidation reaction, and we identified good probes for a model protease DegP. These probes were covalently linked to the C-termini of the cleaved peptides in a mild condition and made the selective enrichment of ligated peptides possible. We suggest that good nucleophilic probes can be found for many other proteases that act via acyl-enzyme intermediates, and these probes will help characterize their substrates.