Inhibition of Class C β-Lactamases:  Structure of a Reaction Intermediate with a Cephem Sulfone^,

Abstract

The crystallographic structure of the Enterobacter cloacae GC1 extended-spectrum class C beta-lactamase, inhibited by a new 7-alkylidenecephalosporin sulfone, has been determined by X-ray diffraction at 100 K to a resolution of 1.6 A. The crystal structure was solved by molecular replacement using the unliganded structure [Crichlow et al. (1999) Biochemistry 38, 10256-10261] and refined to a crystallographic R-factor equal to 0.183 (R(free) 0.208). Cryoquenching of the reaction of the sulfone with the enzyme produced an intermediate that is covalently bound via Ser64. After acylation of the beta-lactam ring, the dihydrothiazine dioxide ring opened with departure of the sulfinate. Nucleophilic attack of a side chain pyridine nitrogen atom on the C6 atom of the resultant imine yielded a bicyclic aromatic system which helps to stabilize the acyl enzyme to hydrolysis. A structural assist to this resonance stabilization is the positioning of the anionic sulfinate group between the probable catalytic base (Tyr150) and the acyl ester bond so as to block the approach of a potentially deacylating water molecule. Comparison of the liganded and unliganded protein structures showed that a major movement (up to 7 A) and refolding of part of the Omega-loop (215-224) accompanies the binding of the inhibitor. This conformational flexibility in the Omega-loop may form the basis of an extended-spectrum activity of class C beta-lactamases against modern cephalosporins.

Hydrolysis of Third-generation Cephalosporins by Class C β-Lactamases

“…In the ES GC1 variant, with the flexible, multiconformation ⍀ loop at the bottom of the binding site, no mutations exist in the 290s loop. Nor is structural disorder in the 290s loop expected, and none has been observed to date (25,26).…”

“…In the ES complex, a quite different orientation is found because the ⍀ loop of the enzyme, longer by a 3-residue insertion, can adopt an alternative folding. This conformational change in the ⍀ loop is presumably initiated by contact between Tyr-221 (numbered 224 in GC1) and an early reaction intermediate, as seen in other GC1 complexes (25,26). The expanded ⍀ loop refolds to a more open conformation so that Tyr-224 moves as much as 10 Å from its WT position, a position now occupied by the methyl group of the oxyimino branch.…”

“…Crowding and Hydrolysis-The inability of the wild-type class C ␤-lactamases to effectively hydrolyze some third-generation cephalosporins has been addressed in structural terms (10,11,26). Not only ligand-protein contacts but also intraligand contacts can reduce the conformational freedom of the acyl intermediate prior to deacylation.…”

Strategic Design of an Effective β-Lactamase Inhibitor

“…A reoriented CϭO group away from the oxyanion hole decreases the rate of deacylation for two reasons: (a) this moiety is no longer primed, via the backbone NH hydrogen bonds in the oxyanion hole, to be receptive for nucleophilic attack by a water; and (b) the carbonyl carbon atom position is now positioned ϳ1.7 Å further away from the deacylation water, a distance likely too great for efficient deacylation by a water molecule. Note that the carbonyl oxygen does not necessarily need to be positioned outside the oxyanion hole to slow down deacylation as observed for a cephem sulfone compound similar to LN-1-255 yielding a bicyclic aromatic intermediate with class C GC-1 ␤-lactamase (43). An additional difference between the LN-1-255 SHV-1 structure and the cephem sulfone class C structure is that the former intermediate is refined, having a planar bicyclic aromatic ring, in accord with the unbiased omit density (Fig.…”

Structure-Based Approach for Binding Site Identification on AmpC β-Lactamase

“…Thus, the structure of AmpC has been determined in complex with inhibitors such as -lactams, 4,[20][21][22] transition state analogues, 23,24 and arylboronic acids, 6,25,26 the leads for which were known before the first structures were determined. 27 There are now 18 inhibitor complex structures with class C -lactamases that have been published.…”