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Carboxylation and Decarboxylation of Active Site Lys 84 Controls the Activity of OXA-24 β-Lactamase of Acinetobacter baumannii: Raman Crystallographic and Solution Evidence
journal contribution
posted on 2012-07-11, 00:00 authored by Tao Che, Robert
A. Bonomo, Sivaprakash Shanmugam, Christopher R. Bethel, Marianne Pusztai-Carey, John D. Buynak, Paul R. CareyThe class D β-lactamases are characterized by the
presence
of a carboxylated lysine in the active site that participates in catalysis.
Found in Acinetobacter baumannii, OXA-24
is a class D carbapenem hydrolyzing enzyme that exhibits resistance
to most available β-lactamase inhibitors. In this study, the
reaction between a 6-alkylidiene penam sulfone inhibitor, SA-1-204,
in single crystals of OXA-24 is followed by Raman microscopy. Details
of its reaction with SA-1-204 provide insight into the enzyme’s
mode of action and help define the mechanism of inhibition. When the
crystal is maintained in HEPES buffer, the reaction is fast, shorter
than the time scale of the Raman experiment. However, when the crystal
holding solution contains 28% PEG 2000, the reaction is slower and
can be recorded by Raman microscopy in real time; the inhibitor’s
Raman bands quickly disappear, transient features are seen due to
an early intermediate, and, at approximately 2–11 min, new
bands appear that are assigned to the late intermediate species. At
about 50 min, bands due to all intermediates are replaced by Raman
signals of the unreacted inhibitor. The new population remains unchanged
indicating (i) that the OXA-24 is no longer active and (ii) that the
decarboxylation of Lys84 occurred during the first reaction cycle.
Using absorbance spectroscopy, a one-cycle reaction could be carried
out in aqueous solution producing inactive OXA-24 as assayed by the
chromogenic substrate nitrocefin. However, activity could be restored
by reacting aqueous OXA-24 with a large excess of NaHCO3, which recarboxylates Lys84. In contrast, the addition of NaHCO3 was not successful in reactivating OXA-24 in the crystalline
state; this is ascribed to the inability to create a concentration
of NaHCO3 in large excess over the OXA-24 that is present
in the crystal. The finding that inhibitor compounds can inactivate
a class D enzyme by promoting decarboxylation of an active site lysine
suggests a novel function that could be exploited in inhibitor design.