Role of a Mutation at Position 167 of CTX-M-19 in Ceftazidime Hydrolysis

Abstract

CTX-M-19 is a recently identified ceftazidime-hydrolyzing extended-spectrum β-lactamase, which differs from the majority of CTX-M-type β-lactamases that preferentially hydrolyze cefotaxime but not ceftazidime. To elucidate the mechanism of ceftazidime hydrolysis by CTX-M-19, the β-lactam MICs of a CTX-M-19 producer, and the kinetic parameters of the enzyme were confirmed. We reconfirmed here that CTX-M-19 is also stable at a high enzyme concentration in the presence of bovine serum albumin (20 μg/ml). Under this condition, we obtained more accurate kinetic parameters and determined that cefotaxime ( k _cat /K _m , 1.47 × 10 ⁶ s ⁻¹ M ⁻¹ ), cefoxitin ( k _cat /K _m , 62.2 s ⁻¹ M ⁻¹ ), and aztreonam ( k _cat /K _m , 1.34 × 10 ³ s ⁻¹ M ⁻¹ ) are good substrates and that imipenem ( k ₊₂ /K , 1.20 × 10 ² s ⁻¹ M ⁻¹ ) is a poor substrate. However, CTX-M-18 and CTX-M-19 exhibited too high a K _m value (2.7 to 5.6 mM) against ceftazidime to obtain their catalytic activity ( k _cat ). Comparison of the MICs with the catalytic efficiency ( k _cat /K _m ) of these enzymes showed that some β-lactams, including cefotaxime, ceftazidime, and aztreonam showed a similar correlation. Using the previously reported crystal structure of the Toho-1 β-lactamase, which belongs to the CTX-M-type β-lactamase group, we have suggested characteristic interactions between the enzymes and the β-lactams ceftazidime, cefotaxime, and aztreonam by molecular modeling. Aminothiazole-bearing β-lactams require a displacement of the aminothiazole moiety due to a severe steric interaction with the hydroxyl group of Ser167 in CTX-M-19, and the displacement affects the interaction between Ser130 and the acidic group such as carboxylate and sulfonate of β-lactams.