Structure of the Extended-Spectrum Class C β-Lactamase of Enterobacter cloacae GC1, a Natural Mutant with a Tandem Tripeptide Insertion,

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Abstract
A class C β-lactamase from a clinical isolate of Enterobacter cloacae strain GC1 with improved hydrolytic activity for oxyimino β-lactam antibiotics has been analyzed by X-ray crystallography to 1.8 Å resolution. Relative to the wild-type P99 β-lactamase, this natural mutant contains a highly unique tandem repeat Ala211-Val212-Arg213 [Nugaka et al. (1995) J. Biol. Chem. 270, 5729−5735]. The 39.4 kDa chromosomal β-lactamase crystallizes from poly(ethylene glycol) 8000 in potassium phosphate in space group P21212 with cell dimensions a = 78.0 Å, b = 69.5 Å, and c = 63.1 Å. The crystal structure was solved by the molecular replacement method, and the model has been refined to an R-factor of 0.20 for all nonzero data from 8 to 1.8 Å. Deviations of model bonds and angles from ideal values are 0.008 Å and 1.4°, respectively. Overlay of α-carbon atoms in the GC1 and P99 β-lactamases results in an rms deviation of 0.6 Å. Largest deviations occur in a loop containing Gln120 and in the Ω loop region (200−218) where the three residues 213−215 are disordered. Possibly as a result of this disorder, the width of the opening to the substrate binding cavity, as measured from the 318−324 β-strand to two loops containing Gln120 and Tyr150 on the other side, is 0.6−1.4 Å wider than in P99. It is suggested that conformational flexibility in the expanded Ω loop, and its influence on adjacent protein structure, may facilitate hydrolysis of oxyimino β-lactams by making the acyl intermediate more open to attack by water. Nevertheless, backbone atoms in core catalytic site residues Ser64, Lys67, Tyr150, Asn152, Lys318, and Ser321 deviate only 0.4 Å (rmsd) from atoms in P99. A rotation of a potential catalytic base, Tyr150, relative to P99 at pH 8, is consistent with the requirement for a lower than normal pKa for this residue.