Small Changes in Hydration Determine Cephalosporinase Activity of OXA-48 β‑Lactamases

β-Lactamase
mediated antibiotic resistance threatens treatment
of bacterial infections. OXA-48 enzymes are clinically important class
D serine β-lactamases (SBLs) that confer resistance to most
β-lactam antibiotics, including carbapenems. However, OXA-48
and related enzymes vary widely in their activity toward different
substrates: OXA-48 primarily hydrolyzes carbapenems, whereas the OXA-163
variant is a cephalosporinase with minimal carbapenemase activity.
The basis of cephalosporinase activity in OXA-163 remains elusive.
Here we use QM/MM reaction simulations (umbrella sampling molecular
dynamics) to study the breakdown of the cephalosporin antibiotic ceftazidime,
a key antibiotic for healthcare-associated infections, by selected
OXA-48 variants. Calculated free energy barriers for ceftazidime deacylation
correctly capture the differing catalytic efficiencies of the studied
enzymes and identify the catalytically competent orientation for bound
ceftazidime. Additionally, we show that high flexibility of the Ω
loop bordering the active site, a determinant of specificity in many
SBLs, is not required for efficient deacylation. Based on our simulations,
cephalosporin breakdown in OXA-163 is efficient due to subtle control
of active site solvation, which requires a particular orientation
of Leu158 in the Ω loop. Our simulations further predict that
a single mutation in the OXA-48 β5-β6 loop (Arg214Ser)
will increase the efficiency of ceftazidime deacylation to that of
OXA-163. The finding that the hydration of the general base in the
active site determines deacylation efficiency is possibly important
in other class D β-lactamases.