Abstract 1645 Exploring the role of post-translational modification on b-lactamase activity of OXA24/40 against carbapenem

Phosphorylation is a crucial post-translational modification (PTM) of antibiotic-resistant pathogens for survival. Even though the phosphor-proteomics studies showed phosphorylation of some b-lactamases, one of the principal mechanisms for antibiotic resistivity, the functional impact, and the atomic level of the modified structures are yet to be explored. We addressed this using NMR and MS studies on a well-known b-lactamases OXA24/40 of Acinetobacter baumannii. OXA24/40 is a SxxK acyl transferase enzyme with a known PTM of carbamylation of lysine (K84 of the active motif of OXA24/40), which is required for the acylation and deacylation of antibiotics in deactivation mechanisms. We revealed that the OXA24/40 can also undergo another PTM at the active site. The 15N-1HN TROSY-based HSQC spectrum, LC-MS spectrum of two species with 80 Daltons mass difference, and resonance at 5.12 ppm in 1D 31P NMR spectrum confirm that the PTM is phosphorylation. Subsequent analysis of the Tandem MS/MS spectrum predicted that the phosphor site of the OXA24/40 could be S81. LC-MS and 1D 31P NMR of S81A reveal that the S81 is the phosphor site. We turned into 13C NMR to understand the impact of phosphorylation on carbamylation. A disappearance of resonance at 164 ppm, relevant to carbamylated lysine, confirms that the phosphorylated WT (pWT) is de-carbamylated. As there is a switch between the PTMs, we analyzed the stability of the enzyme using the DSF by measuring melting temperature. The stability of the pWT was higher than that of unpWT at pH 7.0. Based on pH-dependent stability assays, we showed the possibility of two hydrogen bonds within the active pocket in pWT and one in unpWT, which could lead to increased stability. The MD simulations also supported this. Even though the pWT is a stable species, the activity of the pWT is unknown. We investigated the effect of phosphorylation on the b-lactamase activity against doripenem. A 1D 1H real-time NMR assay involves the continuous recording of the 1H resonance of the peaks of the free substrate and products. The analyzed NMR spectra revealed that the rate of the substrate decay is 5.65 *10-2 ± 0.03 s-1 for the unpWT where no substrate decay for pWT. For further understanding, we analyzed the activity of pWT against imipenem, a smaller carbapenem than doripenem without the 11-CH3 group. Surprisingly, the pWT retained the activity against imipenem with the rate of 2.57 *10-2 ± 0.04 s-1. The activity suggests a possibility of competition between the antibiotic and phosphate for S81. The 31P NMR and 1-D 13C NMR experiment after the addition of imipenem showed a resonance switching from 5.32 ppm of bound phosphate to 1.32 ppm of free phosphate in 1-D 31P NMR, and the appearance of resonance at 164 ppm in 1-D 13C NMR spectrum revealed that the imipenem competitively binds the pWT. Even though the phosphorylation de-carbamylate K84 of OXA24/40, the binding pose of different carbapenems can alter the PTMs and activity of pWT. We speculate that the PTM switch and inactivity of phosphorylation against different carbapenems can lead to understanding the effect of PTM on the activation of b-lactamases in pathogens and the development of effective drugs. This work is supported by NIH 1R01GM123338.