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

Article abstract of DOI:10.1002/anie.201711308

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.

Full text of DOI:10.1002/anie.201711308

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Accepted Article
Title: A New Mechanism for β-Lactamases: Class D Enzymes Degrade
1β-Methyl Carbapenems via Lactone Formation
Authors: Christopher T. Lohans, Emma van Groesen, Kiran Kumar,
Catherine L. Tooke, James Spencer, Robert S. Paton, Jürgen
Brem, and Christopher Schofield
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b
A New Mechanism for -Lactamases: Class D Enzymes Degrade
b
1 -Methyl Carbapenems via Lactone Formation
Christopher T. Lohans,[a] Emma van Groesen,[a] Kiran Kumar,[a] Catherine L. Tooke,[b] James
Spencer,[b] Robert S. Paton,[a] Jürgen Brem,[a] Christopher J. Schofield*[a]
Abstract: b-Lactamases threaten the clinical use of carbapenems,
considered antibiotics of last resort. The classical mechanism of
serine carbapenemase activity proceeds via hydrolysis of an acyl-
enzyme intermediate. We show that class D b-lactamases also
degrade clinically used 1b-methyl-substituted carbapenems via the
unprecedented formation of a carbapenem-derived b-lactone. b-
Lactone formation results from the nucleophilic attack of the
carbapenem hydroxyethyl side chain onto the ester carbonyl of the
acyl-enzyme intermediate. The carbapenem-derived lactone products
inhibit both serine b-lactamases (particularly class D) and metallo-b-
lactamases. These results define a new mechanism for the class D
carbapenemases, in which a hydrolytic water molecule is not required.
product with the pyrroline ring present as the D1 tautomer (Table
S1-S3); we propose that the configuration at C-2 is (S) based on
NOESY spectra and coupling constants (Figure S2, S3).
The hydrolysis of carbapenem antibiotics by b-lactamases (i.e.,
carbapenemases) represents a major threat to clinically important
antibiotics of last resort.[1,2] The classical mechanism of the serine
carbapenemases proceeds via a two-step pathway, involving the
formation of an acyl-enzyme intermediate, which is then
hydrolyzed by a nucleophilic water molecule.[3] All clinically used
carbapenem antibiotics have a 6-hydroxyethyl side chain, the
presence of which is proposed to disrupt the hydrolysis step.[4–6]
We show that the class D b-lactamases employ a previously
unidentified mechanism for carbapenem degradation in which a
b-lactone is formed (Figure 1A). We provide evidence that lactone
formation occurs via a mechanism in which the hydroxyl group of
the hydroxyethyl side chain acts as a nucleophile in place of the
water molecule required for hydrolysis.
While characterizing the carbapenemase activity of the class
D serine b-lactamase (SBL) OXA-48 (arguably one of the most
clinically important carbapenemases)[7] with the carbapenem
ertapenem (Figure S1), we observed two major (and two minor)
products by NMR spectroscopy (Figure 1B). The proton 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) (Figure 1B). Further NMR analyses
indicated that this corresponds to the anticipated hydrolysis
Figure 1. (A) Major carbapenem-derived products formed by b-lactamases. The
C-2 stereochemistry of the products may depend on the particular enzyme. (B)
NMR spectra (600 MHz) showing the different product profiles of ertapenem (1
mM) with NDM-1 (5 µM) or OXA-48 (5 µM). Note that the minor peaks represent
products with an assigned (R) configuration at C-2 (indicated with a prime
symbol; see main text and Figure S2-S5).
Chemical shift assignments for the other major product
differed substantially in the region corresponding to the
hydroxyethyl side chain (Table S4, S5). Further analyses led to
the proposal that this product contains a b-lactone, in which the
oxygen derived from the hydroxyethyl side chain is covalently
bonded to the carbonyl originating from the carbapenem b-lactam
ring (Figure 1A). The chemical shift assignments for this product
resemble those of known b-lactones (Table S6). Like the major
hydrolysis product, the major lactone product is observed as the
D1 tautomer (Table S4, S5), and is proposed to have an (S)-
configuration at C-2 (Figure S4, S5). Notably, b-lactone formation
[a]
[b]
Dr. C. T. Lohans, E. van Groesen, K. Kumar, Prof. Dr. R. S. Paton,
Dr. J. Brem, Prof. Dr. C. J. Schofield
Department of Chemistry
University of Oxford
Oxford, UK, OX1 3TA
E-mail: christopher.schofield@chem.ox.ac.uk
C. L. Tooke, Prof. Dr. J. Spencer
School of Cellular and Molecular Medicine
University of Bristol
Bristol, UK, BS8 1TD
Supporting information for this article is given via a link at the end of
the document.
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was observed when ertapenem was incubated with Escherichia
coli expressing OXA-48 (Figure S6).
the lysine is not carbamylated (e.g., due to low pH or
mutations).[5,15,16]
The ertapenem-derived lactone and hydrolysis products were
isolated by HPLC (Figure S7) and characterized by NMR (Figure
S8-S15) and MS (Figure S16). The infrared spectrum of the
lactone product showed a new peak at 1809 cm-1 (Figure S17),
consistent with a b-lactone carbonyl.[8,9] The lactone 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 (Table S3, S5). The
minor products were assigned as having an (R)-configuration at
C-2, but are otherwise the same as the (2S)-lactone and (2S)-
hydrolysis products (Figure S2-S5).
A time 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 (2S)-lactone product predominates at later time points, the
(2R)-lactone is observed at relatively higher levels at early time
points. Incubation of the purified lactone and hydrolysis products
in buffered D2O showed non-enzymatic interconversion between
the (2S)- and (2R)-diastereomers (Figure S20). Therefore, the
greater amount of the (2S)-diastereomers observed in our initial
spectra (Figure 1B) may reflect their relative thermodynamic
stability, whereas the (2R)-diastereomers may represent a
greater proportion of the nascent enzymatic products.
The generality of b-lactone formation by OXA-48 was next
examined. In addition to ertapenem, b-lactone formation was
observed for meropenem, biapenem, and doripenem (Figure
S21-S24, Table S8-S16). However, no b-lactone formation was
observed for imipenem and panipenem (Figure S25, S26, Table
S17-S20). The product profiles of OXA-10 and OXA-23 with these
carbapenems are similar (Figure S21-S26). Therefore, within
limits of detection, b-lactone formation appears to require the
Figure 2. (A) Proposed outline mechanisms for carbapenem hydrolysis and
lactone formation. KCX labels the carbamylated lysine residue, which acts as a
general base. Lactone formation was only observed for carbapenems bearing
a 1b-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 a particular configuration at
C-2 predominates. (B) DFT non-covalent interaction isosurfaces showing
unfavorable steric interactions between the carbapenem 1b-Me and
hydroxyethyl groups, alleviated in the corresponding 1b-H system. Reduced
density gradient isosurface s= 0.5, r(r) signl2 (e/au) colorscale runs from -0.02
a.u. (blue) to 0.02 a.u. (red). (C) Representations of two major conformations of
the hydroxyethyl side chain observed during MD simulation of the OXA-1
doripenem complex;[12] the upper conformation appears to be favorable for b-
lactone formation. Note that the doripenem thiol side chain and serine backbone
are represented as methyl groups.
presence of
a
1b-methyl substituent. While the MBL
carbapenemases NDM-1, CphA, and L1 demonstrated
carbapenemase activity, no b-lactone formation was observed.
(Figure S21-S26) Similarly, only hydrolysis products were
observed with the class A carbapenemase SFC-1 (Figure S21-
S26).[10]
b-Lactone formation likely results from the intramolecular 4-
exo-trig cyclization[11] of the hydroxyethyl oxygen onto the ester
carbonyl of the acyl-enzyme intermediate (Figure 2A).
Crystallographic studies of OXA-1 with doripenem[12] and of OXA-
58 with
a
6a-hydroxymethyl penicillin[13] indicate that the
catalytically important carbamylated lysine residue interacts with
the hydroxyethyl side chain (Figure S27). Furthermore, the
hydroxyethyl hydroxyl 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 a general base.[12,13] Although this conformation of the
hydroxyethyl side chain was not observed in related crystal
structures (Figure S27), these structures depict enzymes in which
Kinetic studies of OXA enzymes show that the kcat values for
1b-methyl-substituted carbapenems tend to be 10-100 fold less
than for those with a 1b-proton.[17,18] Non-covalent interaction
plots indicate that the 1b-methyl group interacts sterically with the
hydroxyethyl group in the acyl-enzyme complex, which is largely
alleviated in the corresponding 1b-proton system (Figure 2B).
Molecular dynamics simulations (100 ns) of the acyl-enzyme
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complex derived from OXA-1 and doripenem[12] also suggest that
the conformation of the hydroxyethyl group is influenced by the
1b-substituent, with the system bearing a 1b-proton showing more
flexibility (Table S21-S23, Figure S28-S31). Notably, in the MD
simulations, the 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).
As 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 ertapenem-derived
lactone was hydrolyzed by SBLs with carbapenemase activity
(SFC-1, OXA-23, and OXA-48; Figure 3A); by contrast, no lactone
hydrolysis was observed by carbapenemase MBLs (NDM-1,
CphA, and L1; Figure 3A). Acylation of some SBLs, such as OXA-
10, by the ertapenem-derived lactone was observed by MS
(Figure 3B, S34). However, the 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).
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, the acyl-
enzyme complexes of 1b-methyl carbapenems with OXA
enzymes are more resistant to hydrolysis than are those derived
from carbapenems without a 1b-methyl group. The class D b-
lactamases have apparently, at least in part, overcome this
inhibition by catalyzing lactone formation. However, lactone
formation may only represent a competitive pathway if hydrolysis
is disfavoured (i.e., by the 1b-methyl group) explaining why only
hydrolysis was observed for carbapenems without a 1b-methyl
group.
The ertapenem-derived lactone inhibited all of the SBLs and
MBLs tested at high concentrations (Figure 3C). Class D b-
lactamases were inhibited particularly strongly, with IC50 values of
790 nM, 1.2 µM, and 940 nM obtained for OXA-10, OXA-23, and
OXA-48, respectively (Figure 3D, S36). However, the potency of
the lactone was relatively low compared to ertapenem (IC50
values of 1.9 nM, 65 nM, and 10 nM for OXA-10, OXA-23, and
OXA-48, respectively; Figure 3D, S36, S37). The observed
inhibition of some MBLs is interesting, given the lack of clinically
used inhibitors for these enzymes.[19]
The degradation of carbapenems via b-lactone formation
represents
a
new mechanism for the carbapenemases.
Carbapenems are potent inhibitors of many PBPs and SBLs, with
which they rapidly react to form an acyl-enzyme complex; this
complex is stabilized due to disruption of the ‘hydrolytic water’ by
the hydroxyethyl side chain. Thus, the results presented here
suggest that, in the case of clinically important 1b-methyl
carbapenems, the class D b-lactamases may have evolved to
overcome this disruption in part by promoting lactone formation.
Although the lactones represent a type of product inhibition, their
potency is less than that of the parent carbapenems. It will be of
interest to see if other mechanisms for fragmentation of the acyl-
enzyme ester evolve.
Avibactam, recently introduced as the first clinically used non-
b-lactam-containing b-lactamase inhibitor, works via a reversible
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.
In this 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 a novel scaffold for b-lactamase inhibition that, with
optimization, may prove comparable in potency to the
carbapenems.
Acknowledgements
We thank the MRC, IMI ENABLE, the Wellcome Trust, and GSK
Figure 3. (A) Extent of hydrolysis of the ertapenem lactone by a panel of
carbapenemases (1 h, 0.5 mM lactone, 5 µM enzyme). (B) Mass spectra
showing acylation of OXA-10 (1 µM) by ertapenem (100 µM) and ertapenem
lactone (100 µM) after 30 min. (C) Inhibitory activity of ertapenem lactone (1
mM, 0.5 mM, 0.1 mM) against a panel of SBLs and MBLs. (D) Comparison of
inhibitory activity of ertapenem and ertapenem lactone against OXA-10.
Tres Contos for support. C.T.L. thanks the CIHR for
postdoctoral fellowship.
a
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[12] K. D. Schneider, M. E. Karpen, R. A. Bonomo, D. A. Leonard, R. A.
Powers, Biochemistry 2009, 48, 11840.
Keywords: antibiotics • antibiotic resistance • carbapenems •
hydrolases • lactones
[13] S. Pratap, M. Katiki, P. Gill, P. Kumar, D. Golemi-Kotra, Antimicrob.
Agents Chemother. 2015, 60, 75.
[1]
[2]
[3]
A. M. Queenan, K. Bush, Clin. Microbiol. Rev. 2007, 20, 440.
G. Patel, R. A. Bonomo, Front. Microbiol. 2013, 4, 48.
J. H. Jeon, J. H. Lee, J. J. Lee, K. S. Park, A. M. Karim, C. R. Lee, B. C.
Jeong, S. H. Lee, Int. J. Mol. Sci. 2015, 16, 9654.
[14] H. B. Burgi, J. D. Dunitz, E. Shefter, J. Am. Chem. Soc. 1973, 95, 5065.
[15] K. D. Schneider, C. J. Ortega, N. A. Renck, R. A. Bonomo, R. A. Powers,
D. A. Leonard, J. Mol. Biol. 2011, 406, 583.
[16] C. M. June, T. J. Muckenthaler, E. C. Schroder, Z. L. Klamer, Z.
Wawrzak, R. A. Powers, A. Szarecka, D. A. Leonard, Protein Sci. 2016,
25, 2152.
[4]
[5]
[6]
[7]
[8]
[9]
L. Maveyraud, L. Mourey, L. P. Kotra, J.-D. Pedelacq, V. Guillet, S.
Mobashery, J.-P. Samama, J. Am. Chem. Soc. 1998, 120, 9748.
C. A. Smith, N. T. Antunes, N. K. Stewart, M. Toth, M. Kumarasiri, M.
Chang, S. Mobashery, S. B. Vakulenko, Chem. Biol. 2013, 20, 1107.
M. Toth, C. A. Smith, N. T. Antunes, N. K. Stewart, L. Maltz, S. B.
Vakulenko, Acta Crystallogr. Sect. D: Struct. Biol. 2017, 73, 692.
L. Poirel, A. Potron, P. Nordmann, J. Antimicrob. Chemother. 2012, 67,
1597.
[17] A. M. Queenan, W. Shang, R. Flamm, K. Bush, Antimicrob. Agents
Chemother. 2010, 54, 565.
[18] S. Oueslati, P. Nordmann, L. Poirel, J. Antimicrob. Chemother. 2015, 70,
1059.
[19] C. Bebrone, Biochem. Pharmacol. 2007, 74, 1686.
[20] D. E. Ehmann, H. Jahić, P. L. Ross, R. F. Gu, J. Hu, G. Kern, G. K.
Walkup, S. L. Fisher, Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 11663.
[21] R. Guerciolini, Int. J. Obes. Relat. Metab. Disord. 1997, 21 Suppl 3, S12.
[22] N. O. Sykes, S. J. Macdonald, M. I. Page, J. Med. Chem. 2002, 45, 2850.
[23] M. E. Migaud, R. C. Wilmouth, G. I. Mills, G. J. Wayne, C. Risley, C.
Chambers, S. J. Macdonald, C. J. Schofield, Chem. Commun. (Camb)
2002, 1274.
A. A. Tymiak, C. A. Culver, M. F. Malley, J. Z. Gougoutas, J. Org. Chem.
1985, 50, 5491.
Y. Pu, F. M. Martin, J. C. Vederas, J. Org. Chem. 1991, 56, 1280.
[10] F. Fonseca, E. I. Chudyk, M. W. van der Kamp, A. Correia, A. J.
Mulholland, J. Spencer, J. Am. Chem. Soc. 2012, 134, 18275.
[11] J. Baldwin, Ciba Found. Symp. 1978, 85.
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Entry for the Table of Contents
COMMUNICATION
Dr. Christopher T. Lohans, Emma van
Groesen, Kiran Kumar, Catherine L.
Tooke, Prof. Dr. James Spencer, Prof.
Dr. Robert S. Paton, Dr. Jürgen Brem,
Prof. Dr. Christopher J. Schofield*
Page No. – Page No.
Re-cycled: In addition to the classical hydrolytic mechanism of carbapenem
degradation, the class D b-lactamases are shown to employ an additional
mechanism whereby a b-lactone product is formed. These b-lactone products
themselves act as b-lactamase inhibitors, representing a potential new scaffold for
inhibitor and antibiotic development.
A New Mechanism for
Class D Enzymes Degrade 1
Carbapenems via Lactone Formation
b
-Lactamases:
b
-Methyl
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