Lohans, C. T., Chan, H. T. H., Malla, T. R., Kumar, K., Kamps, J. J. A. G.,McArdle, D. J. B., ... Schofield, C. J. (2019). Non-Hydrolytic -LactamAntibiotic Fragmentation by l,d-Transpeptidases and Serine -LactamaseCysteine Variants. Angewandte Chemie - International Edition, 58(7), 1990-1994. https://doi.org/10.1002/anie.201809424

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German Edition: DOI: 10.1002/ange.201809424AntibioticsInternational Edition: DOI: 10.1002/anie.201809424

Non-Hydrolytic b-Lactam Antibiotic Fragmentation by ll,,dd-Transpeptidases and Serine b-Lactamase Cysteine VariantsChristopher T. Lohans+, H. T. Henry Chan+, Tika R. Malla, Kiran Kumar, Jos J. A. G. Kamps,Darius J. B. McArdle, Emma van Groesen, Mariska de Munnik, Catherine L. Tooke,James Spencer, Robert S. Paton, Jgrgen Brem, and Christopher J. Schofield*

Abstract: Enzymes often use nucleophilic serine, threonine,and cysteine residues to achieve the same type of reaction; theunderlying reasons for this are not understood. While bacteriald,d-transpeptidases (penicillin-binding proteins) employa nucleophilic serine, l,d-transpeptidases use a nucleophiliccysteine. The covalent complexes formed by l,d-transpepti-dases with some b-lactam antibiotics undergo non-hydrolyticfragmentation. This is not usually observed for penicillin-binding proteins, or for the related serine b-lactamases.Replacement of the nucleophilic serine of serine b-lactamaseswith cysteine yields enzymes which fragment b-lactams viaa similar mechanism as the l,d-transpeptidases, implying thedifferent reaction outcomes are principally due to the forma-tion of thioester versus ester intermediates. The results highlightfundamental differences in the reactivity of nucleophilic serineand cysteine enzymes, and imply new possibilities for theinhibition of nucleophilic enzymes.

Nature employs enzymes with nucleophilic serine, threo-nine, or cysteine residues to catalyze closely related reactions.Well-known examples of this are the serine and cysteineproteases, in which these different nucleophilic residues areused to cleave peptide bonds. An analogous situation existsfor the transpeptidase enzymes involved in bacterial pepti-doglycan biosynthesis; the d,d-transpeptidases (or penicillin-binding proteins; PBPs) employ a nucleophilic serine,whereas the l,d-transpeptidases (Ldts) employ a nucleophiliccysteine. The reasons for the use of a particular nucleophilicresidue in a particular enzyme context are not understood.

Bacterial peptidoglycan biosynthesis is an antibiotic targetof immense clinical importance.[1–3] Peptidoglycan, an extra-cellular network of polysaccharides and cross-linked peptides,plays a critical structural role in the bacterial cell wall.[4] Inmany Gram-negative bacteria, cross-links occur primarilybetween meso-diaminopimelate (meso-Dap) and d-Ala res-idues. Formation of these meso-Dap-d-Ala cross-links iscatalyzed by the d,d-transpeptidase activities of the PBPs.However, the peptidoglycan of some bacteria (for example,Mycobacterium tuberculosis in stationary phase) consistssubstantially of cross-links formed between meso-Dap resi-dues, catalyzed by the Ldts.[5, 6] The transpeptidase activities ofPBPs and Ldts both proceed via acyl-enzyme substratecomplexes formed by reaction with a donor substrate peptide;the acyl-enzyme complex subsequently reacts with an aminogroup of an acceptor peptide to give the cross-linked product.While the acyl-enzyme complex is formed by reaction witha nucleophilic serine residue in the PBPs, the Ldts insteademploy a nucleophilic cysteine.

The PBPs are established targets of b-lactam drugs, themost widely used class of antibiotic.[7] b-Lactams (Figure 1A)inhibit PBPs by covalently modifying the nucleophilic serine(Figure 1B). The use of b-lactams is compromised bybacterial resistance mechanisms, most importantly the pro-duction of serine b-lactamases (SBLs). The SBL mechanisminvolves the reaction of the b-lactam ring with a nucleophilicserine to form an acyl-enzyme complex (Figure 1 B), which isthen efficiently hydrolyzed. Three of the five clinically usedSBL inhibitors (for example, clavulanic acid) react irrever-sibly to form hydrolytically stable acyl-enzyme complexes,whilst the other two (avibactam and vaborbactam) reactreversibly.[8]

The nucleophilic cysteine of the Ldts can also be acylatedby b-lactams (Figure 1B).[9, 10] In particular, carbapenems andpenems (Figure 1A) are potent Ldt inhibitors,[9, 11] andmanifest antibiotic activity against M. tuberculosis.[12,13] How-ever, the Ldts are often considered insensitive to the otherclasses of b-lactam antibiotics (for example, penicillins).[6, 14,15]

Recent work has suggested that the acyl-enzyme com-plexes formed by some b-lactams with Ldts behave differentlythan the analogous complexes formed with PBPs or SBLs(Figure 1B).[9, 12,16] However, it is unclear whether the differ-ence in reactivity arises from the cysteine-for-serine substitu-tion, or if it is due to other active site variations. Based onstudies of LdtMt2 (from M. tuberculosis) and cysteine variantsof the OXA-48 and KPC-2 SBLs, we report that the differ-ence in reactivity is principally due to the identity of thenucleophilic residue, and arises from the different chemical

[*] Dr. C. T. Lohans,[+] H. T. H. Chan,[+] T. R. Malla, K. Kumar,J. J. A. G. Kamps, D. J. B. McArdle, E. van Groesen, M. de Munnik,Prof. Dr. R. S. Paton, Dr. J. Brem, Prof. Dr. C. J. SchofieldDepartment of Chemistry, University of OxfordOxford, OX1 3TA (UK)E-mail: christopher.schofield@chem.ox.ac.uk

C. L. Tooke, Prof. Dr. J. SpencerSchool of Cellular and Molecular Medicine, University of BristolBristol, BS8 1TD (UK)

[++] These authors contributed equally to this work.

Supporting information and the ORCID identification number(s) forthe author(s) of this article can be found under:https://doi.org/10.1002/anie.201809424.

T 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co.KGaA. This is an open access article under the terms of the CreativeCommons Attribution License, which permits use, distribution andreproduction in any medium, provided the original work is properlycited.

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properties of the ester and thioester intermediates. This isa rare example where the identity of the nucleophilic residuechanges the reaction outcome of an enzyme. The resultssuggest that the biological use of specific nucleophiles may berelated to function, and raise the potential of developing newnucleophilic-enzyme inhibitors (including for serine andcysteine proteases) and antibiotics, i.e., which are poised toundergo fragmentation reactions, yielding stable acyl-enzymecomplexes.

To compare the reactivity of the Ldts with the nucleo-philic serine enzymes (i.e., PBPs and SBLs), mass spectrom-etry (MS) was used to monitor the adducts formed by LdtMt2

with a panel of b-lactam antibiotics. While screening repre-sentative penicillins, mass shifts were observed that areconsistent with the formation of acyl-enzyme complexeswith oxacillin, piperacillin, and ticarcillin, but not withampicillin (Figure 1C and Supporting Information, Fig-ure S1). Over time, a new species 159 Da smaller than theintact acyl-enzyme complexes accumulated for LdtMt2 in thepresence of ampicillin, oxacillin, and piperacillin (Figure 1C,and Supporting Information, Figures S1 and S2). This massdifference is consistent with the cleavage of the C5–C6 bondof the penicillin-derived LdtMt2 acyl-enzyme complex, yield-ing LdtMt2 modified by an acetyl group bearing the penicillinC6 side chain. This is similar to what has been observed

previously for ampicillin with other Ldts.[16, 17] In the case offaropenem, a peak corresponding to LdtMt2 acylated with a 3-hydroxybutyrate fragment was observed (Figure 1D).[9, 12] Asimilar adduct was observed following treatment of LdtMt2

with the penem sulopenem (Supporting Information, Fig-ure S1). Incubation of LdtMt2 with the cephalosporins ceph-alothin, cefotaxime, and cefapirin resulted in adducts in whichthe C3’ leaving groups were lost (Supporting Information,Figure S1). Fragmentation was not observed for the carbape-nems meropenem, imipenem, and ertapenem (SupportingInformation, Figure S1), and acylation was not observed forthe monobactam aztreonam and the oxacephem moxalactam.NMR experiments indicated that cephalothin, meropenem,aztreonam, and moxalactam were not efficiently degraded byLdtMt2 under the MS assay conditions. No acylation of theLdtMt2 C354S variant (where the nucleophilic cysteine isreplaced with a serine) by b-lactams was observed under theconditions used for MS (Supporting Information, Figure S3).

The fragmentation of penicillins and penems with LdtMt2,which is not typically observed for PBPs and SBLs, may resultfrom the different chemical properties of the thioester (Ldts)and ester (PBPs and SBLs) acyl-enzyme complexes. Alter-natively, these differences could arise from other features ofthe active site; for example, there is less than 25% sequenceidentity between PBP5 from Pseudomonas aeruginosa andLdtMt2 from M. tuberculosis (active sites are compared inFigure S4 in the Supporting Information). To investigate,variants of the clinically important OXA-48 and KPC-2 SBLswere prepared in which the nucleophilic serine was replacedwith a cysteine. Consistent with previous reports on thecysteine variants of SBLs,[18–20] OXA-48 S70C (kcat/KM 0.098:0.017 mm@1 s@1 with the fluorogenic cephem substrate FC5)[21]

was not as catalytically active as the wild-type enzyme (kcat/KM 0.36: 0.06 mm@1 s@1 with FC5; Supporting Information,Table S1). However, the impact of this substitution on theenzymatic mechanism of the SBLs has not been previouslydescribed.

Following incubation of the penicillin ampicillin withwild-type OXA-48 or KPC-2, only hydrolysis was observed byNMR (Figure 2A). While OXA-48 S70C and KPC-2 S70Calso catalyzed ampicillin hydrolysis, additional products wereformed (Figure 2A). Based on further NMR and MSanalyses, these products were assigned as the d-phenyl-glycylglycine dipeptide (derived in part from C6 and C7 ofampicillin) and a dihydrothiazole (derived from the ampicillinthiazolidine ring) (Figure 2B, Supporting Information, Figur-es S5 and S6, and Tables S2–S5). The assignment of d-phenylglycylglycine was validated by comparison with a syn-thetic standard (Supporting Information, Figure S7). Treat-ment of other penicillins (for example, piperacillin, oxacillin,and ticarcillin) with OXA-48 S70C and KPC-2 S70C resultedin the formation of both hydrolysis and C5–C6 fragmentationproducts (Figure 2C and Supporting Information, Figure S8).While LdtMt2-catalyzed penicillin fragmentation could not beobserved by NMR (Supporting Information, Figure S8), MSanalyses indicated that OXA-48 S70C and LdtMt2 form thesame C5–C6 fragmentation products (Figure 1C and Sup-porting Information, Figure S9). Although the relative levelsof hydrolysis and C5–C6 fragmentation were similar for both

Figure 1. A) The penicillin, cephalosporin, penem, and carbapenem b-lactam classes. B) Reaction of the nucleophilic serine (PBPs, SBLs) orcysteine (Ldts) with a generic penicillin, forming an acyl-enzymecomplex which can undergo hydrolysis or C5–C6 fragmentation. MSdata for LdtMt2 (4 mm) incubated with a 100-fold excess of C) oxacillinfor 2 h, and D) faropenem for 10 min. Black spectra: LdtMt2 ; bluespectra: LdtMt2 with the indicated b-lactam.

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OXA-48 S70C and KPC-2 S70C, LdtMt2 was observed to favorhydrolysis of the penicillin b-lactam (contrasting with Ldtfm

from Enterococcus faecium, for which fragmentation isreported to predominate).[16]

Treatment of faropenem with OXA-48 S70C or KPC-2S70C yielded different products than the hydrolysis productsformed by wild-type OXA-48 and KPC-2 (Figure 3A andSupporting Information, S10). Based on NMR and MS, theseproducts were assigned as 3-hydroxybutyrate (derived in partfrom C6 and C7 of faropenem) and a thiazole (derived fromthe dihydrothiazole ring of faropenem) (Figure 3 B, Support-ing Information, Figure S10 and Tables S6–S9). Further NMR

analyses demonstrated that the same faropenem-derivedthiazole is formed by LdtMt2 ; enzyme-catalyzed faropenemhydrolysis was not observed for LdtMt2 or OXA-48 S70C(Supporting Information, Figure S11).

The products formed by LdtMt2 and the SBL thiol variantswith penicillins and penems are consistent with C5–C6 bondcleavage, and inform on the mechanism of fragmentation(Figure 4A,B). The penicillin-derived dihydrothiazole andfaropenem-derived thiazole products imply that the nitrogen/sulfur lone pairs in the thiazolidine (for penicillins) ordihydrothiazole (for faropenem) rings promote C5–C6 bondcleavage. Both reaction pathways likely proceed via a thio-ester-enolate intermediate involving the nucleophilic cys-teine. Formation of this thioester-enolate is expected to bemore energetically favorable than the formation of theanalogous ester-enolate (involving a nucleophilic serine). Asthe stability of these enolates appeared to relate to the acidityat the a-position of the corresponding ester/thioester, calcu-lations were performed to compare the pKa values for theester and thioester species derived from b-lactam fragmenta-tion (Supporting Information, Figure S12 and Tables S10–S13). The calculated pKa values for the thioesters derivedfrom ampicillin and faropenem fragmentation (as seen withthe nucleophilic cysteine enzymes) are 7.2 and 6.4 units lower,respectively, than those for the analogous ester species (whichwere not seen for the nucleophilic serine enzymes) (Support-ing Information, Table S11). These calculated values are

Figure 2. Reaction of penicillins with LdtMt2 and SBL thiol variants.A) 1H NMR spectra (700 MHz) of methyl group resonances for prod-ucts formed upon addition of ampicillin to OXA-48 S70C, compared towild-type OXA-48. These signals correspond to methyl groups indi-cated by the asterisks in panel B. B) Scheme showing ampicillin-derived fragmentation products formed by LdtMt2 and SBL thiolvariants, and hydrolysis products formed by wild-type SBLs, SBL thiolvariants, and LdtMt2. C) 1H NMR spectra (600 MHz) showing theformation of the same dihydrothiazole fragmentation product uponincubation of OXA-48 S70C with ampicillin, piperacillin, oxacillin, andticarcillin (see also Figure S8 in the Supporting Information). Note theother signals present arise from intact penicillins and hydrolyzedproducts.

Figure 3. Reaction of faropenem with LdtMt2 and SBL thiol variants.A) 1H NMR spectra (600 MHz) showing the methyl group resonancesof products formed upon addition of faropenem to OXA-48 S70C andOXA-48. Note, the two doublets (d 1.28 ppm, 1.29 ppm) observed forthe faropenem hydrolysis product were assigned as C5 epimers (seeTable S7 in the Supporting Information), although double bond iso-mers are also possible. The highlighted signals correspond to methylgroups indicated by asterisks in panel B. B) Scheme showing the C5–C6 faropenem fragmentation products observed for LdtMt2 and SBLthiol variants, as compared to the hydrolysis products observed forwild-type SBLs.

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consistent with experimentally determined pKa values forethyl acetate (pKa = 25.6)[22] and ethyl thioacetate (pKa =

20.4–21.5).[23] Thus, the favorable formation of thioester-enolates may rationalize why enzymes with nucleophiliccysteine residues (for example, Ldts and SBL thiol variants)catalyze b-lactam C5–C6 fragmentation, while those withnucleophilic serine residues (for example, PBPs and SBLs)typically do not.

Fragmentation was not observed for the acyl-enzymecomplexes derived from carbapenems with LdtMt2 (Fig-ure 4C), contrasting with the rapid fragmentation of thepenems faropenem and sulopenem, suggesting the heteroa-tom at position 1 is important. While the C5–C6 bondcleavage of the faropenem-derived acyl-enzyme complexforms an aromatic thiazole (Figure 4 B), the product imme-diately formed following C5–C6 cleavage of a carbapenem-derived complex would not be aromatic (although it wouldlikely rapidly tautomerize to give an aromatic pyrrole).Furthermore, the pyrroline ring of the carbapenem-derivedacyl-enzyme complex can tautomerize, impacting on theavailability of the b-lactam-derived nitrogen lone pair.Analogous C6–C7 bond cleavage of the cephalosporin-derived acyl-enzyme complexes was also not observed (Fig-

ure 4D). Instead, elimination of the cephalosporin C3’substituent occurs, limiting the ability of the nitrogen lonepair in the dihydrothiazine ring to promote C6–C7 cleavage.

These results imply that the nature of the nucleophilicresidue can determine the fate of the acyl-enzyme complex;however, other active site features can be relevant. As we andothers have observed, the LdtMt2 C354S variant does not formcovalent complexes with the carbapenems and penemstested.[12] Furthermore, the C5–C6 fragmentation of benzyl-penicillin has been described for a PBP from a Streptomycesspp.,[24] while the formation of a thiazole product fromfaropenem was observed for some class C SBLs.[25] It could bethat as yet unidentified features in the active sites of somenucleophilic serine enzymes promote C5–C6 fragmentation.The acyl-enzyme complexes of SBLs with some other b-lactams favor alternative (i.e., not C5–C6) fragmentationreactions, as reported for the SBL inhibitors clavulanic acid,sulbactam, and tazobactam.[7, 8] Alternative enzyme-catalyzedmechanisms not involving C@C fragmentation have also beenobserved, including the degradation of carbapenems byclass D SBLs via lactone formation.[26]

There are few examples where substitution of thenucleophilic residue determines the type of reaction catalyzedby an enzyme. While the wild-type protease subtilisincatalyzes hydrolysis, the subtilisin S221C variant also mani-fests transpeptidase activity.[27] More typically, substitution ofa nucleophilic serine with cysteine can stabilize the acyl-enzyme complex.[28, 29] However, acylation by the naturalsubstrate may not occur following the substitution of serinewith cysteine (and vice versa), as observed by us (for LdtMt2

C354S) and others.[30, 31]

The Ldts are antibiotic targets in pathogens, notably M.tuberculosis.[6,12] These results indicate that the differentreactivities of the Ldts with b-lactam antibiotics, as comparedto the PBPs and SBLs, are substantially governed by thenucleophilic cysteine, which facilitates fragmentation reac-tions not normally observed for enzymes employing nucleo-philic serines. These fragmentation reactions can be ration-alized mechanistically by the relatively favorable involvementof a thioester-enolate intermediate in fragmentation. Theseresults highlight a potential new approach for the develop-ment of antibiotics targeting the Ldts, for example, theidentification of b-lactams that undergo C5–C6 (or C6–C7 forcephalosporins) fragmentation to give stable acyl-enzymecomplexes. They may also inspire the development of relatedinhibitors targeting other classes of enzymes with nucleophiliccysteine residues (for example, the cysteine proteases). Thepotential for toxic and/or immunomodulatory effects of the b-lactam fragments/complexes formed by the Ldts should alsobe considered.[32]

More generally, these results suggest that it is of interest toconsider links between the biological roles of nucleophilicenzymes and the nature of the nucleophilic residue employed,including in the many biologically important serine/cysteineproteases and related enzymes (for example, those involvedin ubiquitin biology).[33] The enhanced nucleophilicity ofthiols compared to alcohols, and the increased reactivity ofthioesters versus esters are often cited as factors for the use ofthiols in biological systems. Our results, combined with prior

Figure 4. Comparison of the reactivities of the acyl-enzyme complexesformed from LdtMt2 with different b-lactam antibiotic sub-classes. Forthe A) penicillins and B) penems, C5–C6 fragmentation gives rise toa thioester-enolate. Note that penicillin hydrolysis occurs preferentiallyto fragmentation for LdtMt2 (Figure 2 and Supporting Information,Figure S8), while only fragmentation of faropenem was detected(Figure 3 and Supporting Information, Figure S11). C) C5–C6 fragmen-tation was not observed for the carbapenems (Supporting Information,Figure S1), potentially in part due to delocalization of the pyrrolinenitrogen lone pair or due to tautomerization of the pyrroline ring (aswell as the lack of a heteroatom at position 1 of the ring). Note thehypothetical product would be expected to tautomerize to a pyrrole.D) C6–C7 fragmentation was not observed for cephalosporins; how-ever, elimination of the C3’ leaving group occurs (Supporting Informa-tion, Figure S1). Note that carbapenem and cephalosporin hydrolysisby LdtMt2 was not observed under our NMR conditions.

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studies on the nucleophilic residues of PBPs and SBLs, implythat both the reaction rate (including if the reaction occurs ornot), and the fate of the acyl-enzyme complex depend on theidentity of the nucleophile and the context in which the esteror thioester exists. Thus, the use of nucleophilic serine orcysteine residues may reflect chemical evolutionary pressuressubject to their biological roles and the types of reactionscatalyzed. The latter may include factors relating to compet-ing pathways/by-products, which the present work shows candiffer with the different types of nucleophile.

Acknowledgements

This project was co-funded by the Tres Cantos Open LabFoundation (Project TC 241). We thank the Wellcome Trust,the Biotechnology and Biological Sciences Research Council(BBSRC) [grant number BB/M011224/1], and the MedicalResearch Council (MRC) for funding our work on antibiotics.

Conflict of interest

The authors declare no conflict of interest.

Keywords: antibiotic resistance · fragmentation · hydrolases ·transpeptidases · b-lactamases

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Manuscript received: August 15, 2018Accepted manuscript online: December 20, 2018Version of record online: January 21, 2019

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