A New Mechanism for β‐Lactamases: Class D Enzymes Degrade 1β‐Methyl Carbapenems through Lactone Formation

Abstract β‐Lactamases threaten the clinical use of carbapenems, which are considered antibiotics of last resort. The classical mechanism of serine carbapenemase catalysis proceeds through hydrolysis of an acyl‐enzyme intermediate. We show that class D β‐lactamases also degrade clinically used 1β‐methyl‐substituted carbapenems through the unprecedented formation of a carbapenem‐derived β‐lactone. β‐Lactone formation results from nucleophilic attack of the carbapenem hydroxyethyl side chain on the ester carbonyl of the acyl‐enzyme intermediate. The carbapenem‐derived lactone products inhibit both serine β‐lactamases (particularly class D) and metallo‐β‐lactamases. These results define a new mechanism for the class D carbapenemases, in which a hydrolytic water molecule is not required.

The hydrolysis of carbapenem antibiotics by b-lactamases (i.e., carbapenemases) represents am ajor threat to clinically important antibiotics of last resort. [1,2] Thec lassical mechanism of the serine carbapenemases proceeds through at wostep pathway involving the formation of an acyl-enzyme intermediate,w hich is then hydrolyzed by an ucleophilic water molecule. [3] All clinically used carbapenem antibiotics have a6 -hydroxyethyl side chain, the presence of which is proposed to disrupt the hydrolysis step. [4][5][6] We show that the class D b-lactamases employ apreviously unidentified mechanism for carbapenem degradation in which a b-lactone is formed ( Figure 1A). We provide evidence that lactone formation occurs through amechanism in which the hydroxy group of the hydroxyethyl side chain acts as anucleophile in place of the water molecule required for hydrolysis.
While characterizing the carbapenemase activity of the class Ds erine b-lactamase (SBL) OXA-48 (arguably one of the most clinically important carbapenemases) [7] with the carbapenem ertapenem ( Figure S1 in the Supporting Information), we observed two major (and two minor) products by NMR spectroscopy ( Figure 1B). Theproton spectrum of one major product was identical to that observed from the hydrolysis of ertapenem by the metallo-b-lactamase (MBL) New Delhi MBL-1 (NDM-1;F igure 1B). Further NMR analyses indicated that this corresponds to the anticipated hydrolysis product with the pyrroline ring present as the D 1 tautomer (Table S1-S3 in the Supporting Information);w e propose that the configuration at C-2 is S based on NOESY spectra and coupling constant analysis ( Figures S2, S3). Chemical shift assignments for the other major product differed substantially in the region corresponding to the hydroxyethyl side chain (Tables S4, S5). Further analyses led to the proposal that this product contains a b-lactone,inwhich the oxygen derived from the hydroxyethyl side chain is covalently bonded to the carbonyl originating from the carbapenem b-lactam ring ( Figure 1A). Thec hemical shift assignments for this product resemble those of known blactones (Table S6). Like the major hydrolysis product, the major lactone product is observed as the D 1 tautomer (Tables S4, S5), and is proposed to have an S configuration at C-2 ( Figures S4, S5). Notably, b-lactone formation was observed when ertapenem was incubated with Escherichia coli expressing OXA-48 ( Figure S6).
Thee rtapenem-derived lactone and hydrolysis products were isolated by HPLC ( Figure S7) and characterized by NMR ( Figures S8-S15) and MS ( Figure S16). Thei nfrared spectrum of the lactone product showed an ew peak at 1809 cm À1 ( Figure S17), which is consistent with a b-lactone carbonyl. [8,9] Thel actone product also behaved as expected with respect to reaction with nucleophiles ( Figure S18, Table S7).
In addition to these major products,lower levels of other species were observed in the reaction mixture ( Figure 1B). Further NMR analyses led to their assignment as stereoisomers of the major lactone and hydrolysis products (Tables S3, S5). Them inor products were assigned as having an R configuration at C-2, but are otherwise the same as the 2S-configured lactone and hydrolysis products (Figures S2-S5).
At ime-course analysis monitoring the relative levels of the ertapenem OXA-48 products suggested that both lactone and hydrolysis products are formed enzymatically (Figure S19). While the assigned 2S-configured lactone product predominates at later time points,the assigned 2R-configured lactone is observed at relatively higher levels at early time points.I ncubation of the purified lactone and hydrolysis products in buffered D 2 Os howed non-enzymatic interconversion between the 2S and 2R diastereomers ( Figure S20). Therefore,t he greater amount of the 2S diastereomers observed in our initial spectra ( Figure 1B)m ay reflect their relative thermodynamic stabilities,whereas the 2R diastereomers may represent ag reater proportion of the nascent enzymatic products.
Theg enerality of b-lactone formation by OXA-48 was next examined. In addition to ertapenem, b-lactone formation was observed for meropenem, biapenem, and doripenem ( Figures S21-S24, Tables S8-S16). However,n ob-lactone formation was observed within detection limits for imipenem and panipenem ( Figures S25, S26, Tables S17-S20). Theproduct profiles of OXA-10 and OXA-23 with these carbapenems are similar (Figures S21-S26). Therefore,w ithin the limits of detection, b-lactone formation appears to require the presence of a1 b-methyl substituent. While the MBL carbapenemases NDM-1, CphA, and L1 demonstrated carbapenemase activity,n ob-lactone formation was observed (Figures S21-S26). Similarly,o nly hydrolysis products were observed with the class Ac arbapenemase SFC-1 (Figures S21-S26). [10] b-Lactone formation likely results from intramolecular 4exo-trig cyclization [11] of the hydroxyethyl oxygen onto the ester carbonyl of the acyl-enzymei ntermediate (Figure 2A). Crystallographic studies of OXA-1 with doripenem [12] and of OXA-58 with a6 a-hydroxymethyl penicillin [13] indicate that the catalytically important carbamylated lysine residue interacts with the hydroxyethyl side chain ( Figure S27). Further-more,the hydroxyethyl hydroxy group can adopt an orientation suitable for nucleophilic attack onto the ester carbonyl (i.e., the Bürgi-Dunitz trajectory), [14] with the carbamylated lysine apparently positioned to act as ag eneral base. [12,13] Although this conformation of the hydroxyethyl side chain was not observed in related crystal structures ( Figure S27), Figure 2. A) Proposed outline mechanisms for carbapenem hydrolysis and lactone formation. KCX labels the carbamylated lysine residue, which acts as ageneral base. Lactone formation was only observed for carbapenemsb earing a1 b-methyl group (blue). The timing of the imine tautomerization is not defined, but likely occurs (in part) prior to fragmentation of the acyl-enzyme complex. It is unclear based on the products observed whether aparticular configuration at C-2 predominates. B) DFT non-covalent interaction isosurfaces showing unfavorable steric interactions between the carbapenem 1b-Me and hydroxyethyl groups, which are alleviated in the corresponding 1b-H system. Reduced density gradient isosurface s = 0.5, 1(r)signl 2 (e/au) color scale runs from À0.02 (blue) to 0.02 (red). C) Representations of two major conformations of the hydroxyethyl group observed during MD simulation of the OXA-1 doripenem complex; [12] the upper conformation appears to be more favorable for b-lactone formation. Note that the doripenem thioether side chain and serine backbone are represented as methyl groups.
these structures depict enzymes in which the lysine is not carbamylated (e.g., due to low pH or mutations). [5,15,16] Kinetic studies of OXA enzymes show that the k cat values for 1b-methyl-substituted carbapenems tend to be 10-100fold less than for those with a1 b-proton. [17,18] Non-covalent interaction plots indicate that the 1b-methyl group may interact sterically with the hydroxyethyl group in the acylenzyme complex, which is largely alleviated in the corresponding 1b-proton system ( Figure 2B). Molecular dynamics simulations (100 ns) of the acyl-enzyme complex derived from OXA-1 and doripenem [12] also suggest that the conformation of the hydroxyethyl group is influenced by the 1bsubstituent, with the system bearing a1 b-proton showing more flexibility (Tables S21-S23, Figures S28-S31). Notably, in the MD simulations,t he 1b-methyl system initially alternates between two conformations ( Figure 2C), one of which appears to be consistent with that expected for b-lactone formation ( Figure S27).
We propose that the 1b-methyl group destabilizes the conformation(s) of the hydroxyethyl side chain required for hydrolysis of the acyl-enzyme complex. Consequently,t he acyl-enzyme complexes of 1b-methyl carbapenems with OXA enzymes are more resistant to hydrolysis than are those derived from carbapenems without a1 b-methyl group.T he class D b-lactamases have apparently,a tl east in part, overcome this inhibition by catalyzing lactone formation. However,l actone formation may only represent ac ompetitive pathway if hydrolysis is disfavoured (i.e., by the 1b-methyl group), thus explaining why only hydrolysis is observed for carbapenems without a1 b-methyl group.
Since the lactone products are isomeric with b-lactams,it was considered that they may interact with b-lactamases and penicillin-binding proteins (PBPs). Indeed, the ertapenemderived lactone was hydrolyzed by SBLs with carbapenemase activity (SFC-1, OXA-23,a nd OXA-48;F igure 3A); by contrast, no lactone hydrolysis was observed with carbapenemase MBLs (NDM-1, CphA, and L1;F igure 3A). Acylation of some SBLs,s uch as OXA-10, by the ertapenemderived lactone was observed by MS ( Figure 3B, Figure S34). However,t he lactone did not show antibiotic activity at the levels tested against E. coli ( Figure S35), nor was acylation observed for the non-essential PBP-5 from E. coli (Figure S34).
Thee rtapenem-derived lactone inhibited both SBLs and MBLs when tested at high concentrations ( Figure 3C). Class D b-lactamases were inhibited particularly strongly, with IC 50 values of 790 nm,1 .2 mm,a nd 940 nm obtained for OXA-10, OXA-23,a nd OXA-48, respectively ( Figure 3D, Figure S36). However,t he potencyo ft he lactone was relatively low compared to ertapenem (IC 50 values of 1.9 nm,6 5nm,a nd 10 nm for OXA-10, OXA-23,a nd OXA-48, respectively;F igure 3D,F igures S36, S37). Theo bserved inhibition of some MBLs is interesting, given the lack of clinically used inhibitors for these enzymes. [19] Thed egradation of carbapenems through b-lactone formation represents anew mechanism for the carbapenemases. Carbapenems are potent inhibitors of many PBPs and SBLs, with which they rapidly react to form an acyl-enzyme complex;t his complex is stabilized due to disruption of the "hydrolytic water" by the hydroxyethyl side chain. Thus,t he results presented here suggest that, in the case of clinically important 1b-methyl carbapenems,t he class D b-lactamases may have evolved to overcome this disruption, in part by promoting lactone formation. Although the lactones represent atype of product inhibition, their potency is less than that of the parent carbapenems.I tw ill be of interest to see whether other mechanisms for fragmentation of the acylenzyme ester evolve.
Avibactam, which was recently introduced as the first clinically used non-b-lactam-containing b-lactamase inhibitor, works through ar eversible acylation mechanism. [20] The observation that carbapenem-derived lactones acylate serine b-lactamases suggests that new classes of b-lactamase inhibitors based on lactones are possible.I nt his regard, it is notable that the b-lactone orlistat is an important medicine, [21] and that lactone antibiotics (e.g.,obafluorin) [8] and inhibitors of serine proteases (and lactam derivatives thereof) [22,23] have been reported. Carbapenem-derived b-lactones thus represent an ovel scaffold for b-lactamase inhibition that, with optimization, may prove comparable in potencyt ot he carbapenems.