Critical involvement of a carbamylated lysine in catalytic function of class D beta-lactamases

beta -Lactamases are the resistance enzymes for beta -lactam antibiotics, o f which four classes are known. beta -lactamases hydrolyze the beta -lactam moieties of these antibiotics, rendering them inactive. It is shown herein that the class D OXA-10 beta -lactamase depends critically on an unusual c arbamylated lysine as the basic residue for both the enzyme acylation and d eacylation steps of catalysis. The formation of carbamylated lysine is reve rsible. Evidence is presented that this enzyme is dimeric and carbamylated in living bacteria. High-resolution x-ray structures for the native enzyme were determined at pH values of 6.0, 6.5, 7.5, and 8.5. Two dimers are pres ent per asymmetric unit. One monomer in each dimer was carbamylated at pH 6 .0, whereas ail four monomers were fully carbamylated at pH 8.5. At the int ermediate pH values, one monomer of each dimer was carbamylated, and the ot her showed a mixture of carbamylated and non-carbamylated lysines. It would appear that, as the pH increased for the sample, additional lysines were " titrated" by carbamylation. A handful of carbamylated lysines are known fro m protein crystallographic data, all of which have been attributed roles in structural stabilization (mostly as metal ligands) of the proteins. This p aper reports a previously unrecognized role for a noncoordinated carbamylat e lysine as a basic residue involved in mechanistic reactions of an enzyme, which indicates another means for expansion of the catalytic capabilities of the amino acids in nature beyond the 20 common amino acids in developmen t of biological catalysts.