Kinetic characterization of GES-22 β-lactamase harboring the M169L clinical mutation

Abstract

The class A β-lactamase GES-22 has been identified in Acinetobacter baumannii isolates in Turkey, and subsequently shown to differ from GES-11 by a single substitution (M169L). Because M169 is part of the omega loop, a structure that is known to have major effects on substrate selectivity in class A β-lactamases, we expressed, purified and kinetically characterized this novel variant. Our results show that compared with GES-11^{6 × His}, GES-22^{6 × His} displays more efficient hydrolysis of penicillins, and aztreonam, but a loss of efficiency against ceftazidime. In addition, the M169L substitution confers on GES-22 more efficient hydrolysis of the mechanistic inhibitors clavulanic acid and sulbactam. These effects are highly similar to other mutations at the homologous position in other class A β-lactamases, suggesting that this methionine has a key structural role in aligning active site residues and in substrate selectivity across the class.

Figure 1. Overall structure of GES-1

The conserved element 1 (S70-K73) is in magenta, conserved element number 2 (S130-N132) is in yellow, conserved element number 3 (K234-G236) is in orange and the omega loop (residues E166, P167, M169 and G170) is in green. G243, T237, E104 and G170 are important residues in substrate selectivity of GES-type β-lactamases. (PDB: 3V3R)

Figure 2. Class A β-lactamase sequence alignment

Various class A β-lactamases were aligned using ClustalW, and the portion of the proteins surrounding M169 is shown.

Figure 3. Superposition of GES-11 (PDB: 3V3R; green) with the class A β-lactamase Toho-1 E166A (PDB 1IYO; cyan) in complex with cefotaxime (magenta)

Residues P167, N170, S237, D240, and R274 create a binding site for the bulky thiazolidine side chain of cefotaxime in Toho-1 E166A structure complex.

Figure 4. The hydrophobic environment around M169

The side-chain of M169 makes contacts with F72, M68, the γ-carbon of E166 and the β-carbon of D176. This group of side-chains forms a hydrophobic core directly underneath the omega loop.