The different inhibition mechanisms of OXA-1 and OXA-24 β-lactamases are determined by the stability of active site carboxylated lysine.
The catalytic efficiency of class D β-lactamases depends critically on an unusual carboxylated lysine as the general base residue for both the acylation and deacylation steps of the enzyme. Microbiological and biochemical studies on the class D β-lactamases OXA-1 and OXA-24 showed that the two enzymes behave differently when reacting with two 6-methylidene penems (penem 1 and penem 3): the penems are good inhibitors of OXA-1 but act more like substrates for OXA-24. UV difference and Raman spectroscopy revealed that the respective reaction mechanisms are different. The penems form an unusual intermediate, a 1,4-thiazepine derivative in OXA-1, and undergo deacylation followed by the decarboxylation of Lys-70, rendering OXA-1 inactive. This inactivation could not be reversed by the addition of 100 mM NaHCO3. In OXA-24, under mild conditions (enzyme:inhibitor = 1:4), only hydrolyzed products were detected, and the enzyme remained active. However, under harsh conditions (enzyme:inhibitor = 1:2000), OXA-24 was inhibited via decarboxylation of Lys-84; however, the enzyme could be reactivated by the addition of 100 mM NaHCO3. We conclude that OXA-24 not only decarboxylates with difficulty but also recarboxylates with ease; in contrast, OXA-1 decarboxylates easily but recarboxylates with difficulty. Structural analysis of the active site indicates that a crystallographic water molecule may play an important role in carboxylation in OXA-24 (an analogous water molecule is not found in OXA-1), supporting the suggestion that a water molecule in the active site of OXA-24 can lower the energy barrier for carboxylation significantly.
From the Departments of Biochemistry.
Tao
T
Che
Christopher R
CR
Bethel
Marianne
M
Pusztai-Carey
Robert A
RA
Bonomo
Paul R
PR
Carey
D040981:Acinetobacter baumannii / Q000201:enzymology* / Q000235:genetics; D000900:Anti-Bacterial Agents / Q000737:chemistry; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000737:chemistry; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008239:Lysine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000077731:Meropenem; D013845:Thienamycins / Q000737:chemistry; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D013845:Thienamycins; D065093:beta-Lactamase Inhibitors; C039404:beta-lactamase OXA-2; D001618:beta-Lactamases; C410345:beta-lactamase OXA-24; D000077731:Meropenem; D008239:Lysine
Carboxylated Lysine; Crystallography; Enzyme Mechanisms; OXA-1; OXA-24; Penems; Physical Methods; Raman Spectroscopy; UV Spectroscopy