pubs.acs.org/Biochemistry Published on Web 11/17/2009 r 2009 American Chemical Society

11840 Biochemistry 2009, 48, 11840–11847

DOI: 10.1021/bi901690r

The 1.4 A Crystal Structure of the Class D β-Lactamase OXA-1 Complexed withDoripenem†,‡

Kyle D. Schneider,§ Mary E. Karpen,§ Robert A. Bonomo, ) David A. Leonard,*,§ and Rachel A. Powers§

§Department of Chemistry, Grand Valley State University, Allendale, Michigan 49401 and )Research Service, Louis Stokes ClevelandDepartment of Veterans Affairs Medical Center, and Department of Pharmacology, Molecular Biology and Microbiology, Case

Western Reserve University School of Medicine, Cleveland, Ohio 44106

Received September 28, 2009; Revised Manuscript Received November 16, 2009

ABSTRACT: The clinical efficacy of carbapenem antibiotics depends on their resistance to the hydrolytic actionof β-lactamase enzymes. The structure of the class D β-lactamase OXA-1 as an acyl complex with thecarbapenem doripenem was determined to 1.4 A resolution. Unlike most class A and class C carbapenemcomplexes, the acyl carbonyl oxygen in the OXA-1-doripenem complex is bound in the oxyanion hole.Interestingly, no water molecules were observed in the vicinity of the acyl linkage, providing an explanationfor why carbapenems inhibit OXA-1. The side chain amine of K70 remains fully carboxylated in the acylstructure, and the resulting carbamate group forms a hydrogen bond to the alcohol of the 6R-hydroxyethylmoiety of doripenem. The carboxylate attached to the β-lactam ring of doripenem is stabilized by a salt bridgeto K212 and a hydrogen bond with T213, in lieu of the interaction with an arginine side chain found in mostother β-lactamase-β-lactam complexes (e.g., R244 in the class A member TEM-1). This novel set ofinteractions with the carboxylate results in a major shift of the carbapenem’s pyrroline ring compared to thestructure of the same ring in meropenem bound to OXA-13. Additionally, bond angles of the pyrroline ringsuggest that after acylation, doripenem adopts the Δ1 tautomer. These findings provide important insightsinto the role that carbapenems may have in the inactivation process of class D β-lactamases.

Carbapenems are broad spectrum β-lactam antibiotics thatrepresent one of the last lines of defense against severe bacterialinfections. Doripenem (Figure 1A) was approved for use in theUnited States in 2007 with indications for urinary tract infec-tions and complicated intra-abdominal infections (1). Though abroad spectrum antibiotic, it has exhibited a higher efficacythan imipenem and meropenem in troublesome Gram-negativespecies such as Pseudomonas aeruginosa and Acinetobacterbaumannii (1).

One of the defining features of carbapenems is the presence ofa 6R-hydroxyethyl moiety in lieu of the more bulky 6β amide sidechains found in other β-lactam antibiotics. This unique structuralelement is thought to be responsible for resistance of these drugsto the hydrolytic action of some β-lactamases (2, 3). Class A, C,and D β-lactamases use a serine nucleophile-based covalentcatalysis strategy (4). Interestingly, carbapenems are able to bindin the active site of these enzymes and can even proceed to a

covalent acyl intermediate with the active site serine (2, 3, 5). Inmany of these β-lactamases however, the water-dependent de-acylation process is impaired. The end result is that carbapenemsserve as effective irreversible inhibitors of many β-lactamases.

The interaction of carbapenems with β-lactamases has beenstudied extensively. X-ray crystallographic studies of imipenemand meropenem bound to class A (3, 6-9), class C (2), and classD (5) enzymes have yielded a wealth of structural detail abouthow the acyl intermediate interacts with active site elements. Inthe case of most class A and class C enzymes, acylation isaccompanied by a conformational change in the carbapenemspecies in which the acyl carbonyl is flipped∼180� away from theoxyanion hole (2, 3, 8). Removal of the carbonyl oxygen from theoxyanion hole explains the lack of carbapenem deacylation. Thisconformational flip was shown to be due to a steric clash betweenthe hydroxyethyl group of the carbapenem and the side chain ofN132 in TEM-1 (7). The substitution of N132 with alanine in thisenzyme allows the acyl intermediate to rotate back into theoxyanion hole, though this alone is not enough to impartdeacylation activity. The acyl carbonyl of meropenem is foundin the oxyanion hole of one class A member, the BlaCβ-lactamase from Mycobacterium tuberculosis (9). This may beexplained by the fact that BlaC has a glycine at the positionhomologous to that of the asparagine typically found in class Aand C enzymes.

Structures of OXA-13 with imipenem and meropenem repre-sent the only class D β-lactamase-carbapenem structures deter-mined so far (5). One feature unique to class D enzymes is thepresence of a carboxylated lysine in the active site that playsthe role of a general base during the catalytic cycle (10-14).

†This research was supported by National Institutes of Health Grant1R15AI082416-01 (D.A.L.). The Veterans Affairs Merit Review Pro-gram,Geriatric Research Education andClinical Care VISN 10, and theNational Institutes of Health (Grant RO1 AI072219) supported R.A.B.Use of the Advanced Photon Source was supported by the U.S.Department of Energy, Office of Science, Office of Basic EnergySciences, under Contract DE-AC02-06CH11357. Use of LS-CATSector 21 was supported by the Michigan Economic DevelopmentCorp. and the Michigan Technology Tri-Corridor for the support ofthis research program (Grant 085P1000817).

‡Coordinates have been deposited in the Protein Data Bank (PDB) asentry 3ISG.*To whom correspondence should be addressed: Department of

Chemistry, Grand Valley State University, 346 Padnos Hall, Allendale,MI 49401. Phone: (616) 331-3241. Fax: (616) 331-3230. E-mail:leonardd@gvsu.edu.

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Interestingly, no carboxylation modification of the active sitelysine was observed in either the apo or meropenem-boundstructures of OXA-13 (5). However, the OXA-13 crystals weregrown at pH 5.5, and pH values of <7 are deleterious towardlysine carboxylation in both OXA-10 (10) and OXA-1 (11). Theester carbonyl oxygen of meropenem is located in the oxyanionhole of OXA-13, so the lack of a properly positioned deacylatingwater molecule was cited as the cause of the inhibitory action ofthat drug.

One feature that is common to all β-lactamase-carbapenemstructures determined so far is the formation of a salt bridgebetween the carboxylate of the drug and a widely conservedarginine residue (e.g., R244 in TEM-1) (3). This arginine is notubiquitous, however, so it is expected that those enzymes lackingthis arginine (such as members of the classD β-lactamase OXA-1subfamily)must use a differentmechanism to attract and stabilizethe carboxylate.

Another feature of the carbapenems is the presence of a doublebond in the pyrroline ring which makes possible a tautomeriza-tion upon cleavage of the β-lactam amide bond (Figure 1B).Several studies have suggested that the Δ1 pyrroline form ofimipenem (bound to TEM-1) andmeropenem (bound to SHV-1)is favored in the acyl-enzyme form (6, 8, 15). It has also beensuggested that the two tautomeric states of the acyl intermediatediffer with regard to their propensity for deacylation versusinhibition (6, 15).

Currently, the mechanism by which carbapenems inhibit classD β-lactamases remains unclear.What are the active site elementsthat determine whether the acyl carbonyl oxygen flips out of theoxyanion hole (as in TEM-1, SHV-1, and AmpC) or remainsinside (as with OXA-13 and BlaC)? After acylation, are watermolecules present in a position appropriate for deacylation? Isthe active site carboxy-lysine involved in binding the drug, or isthismodification lost upon acylation?Does tautomerization playany role in the inhibition process, as suggested for the class Aenzymes? To explore these issues, we determined the crystal

structure of the class D β-lactamase OXA-1 complexed withdoripenem to 1.4 A resolution. Beyond illuminating the interac-tions of carbapenems and class D β-lactamases, this structure isthe first to show doripenem bound to a β-lactamase and the firstligand-bound structure of any member of the OXA-1 subfamily.

MATERIALS AND METHODS

Expression and Purification. Escherichia coli cells expres-sing the blaoxa-1 genewere harvested, and the enzymewas purifiedby carboxy-methyl cellulose CM-32 ion-exchange chromatogra-phy (13). OXA-1 purity was estimated to be >95% bySDS-PAGE.1 Pure enzyme was concentrated (Amicon Ultra)to the OXA-1 saturation limit (∼6.0 mg/mL) in preparation forcrystallization.Crystal Growth and Structure Determination. OXA-1

β-lactamase was crystallized as described previously (14). Briefly,a 10 μL drop containing 3.0 mg/mL OXA-1, 10% (v/v) poly-ethylene (PEG) 8000, and 50 mM HEPES (pH 7.5) wasequilibrated by hanging drop vapor diffusion over 20% (v/v)PEG 8000 and 100 mM HEPES (pH 7.5) using microseedingtechniques. Crystals appeared within 2-3 days at 25 �C withmaximal growth (0.05mm� 0.05 mm� 0.15 mm) achieved after1 week. Diffraction data were collected on the LS-CAT beamline(21-ID-D) at the Advanced Photon Source (Argonne, IL) at100 K using a MarCCD detector. Prior to data collection, thecrystal was soaked in a solution containing >20 mM doripenemfor 3 min and then transferred into well buffer containing 25%2-methyl-2,4-pentanediol (MPD) for 30 s before being flash-cooled in liquid nitrogen. A single crystal was used to collect bothhigh- and low-resolution data sets.

Reflections were indexed, integrated, and scaled usingHKL2000 (16). The space group was P21 with two OXA-1monomers in the asymmetric unit (Table 1). The structure ofthe OXA-1-doripenem complex was determined with Pha-ser (17) using the A monomer of apo OXA-1 (PDB entry1M6K) as the initial phasing model (all water and ligandmolecules removed). Refinement and electron density mapcalculations were performed with Refmac5 (18) in the CCP4Program Suite (19). Manual rebuilding of the model was donewith Coot (20). The final model of the OXA-1-doripenemcomplex contained 249 residues in each monomer, 434 watermolecules, and one molecule of MPD. Doripenem was modeledinto each active site on the basis of initial Fo - Fc differenceelectron density maps and further refined with Refmac5. Thecoordinates for this structure have been deposited in the ProteinData Bank as entry 3ISG.Molecular Modeling. Starting coordinates for the semi-

empirical molecular modeling of doripenem were obtained froman earlymodel of theOXA-1-doripenem crystal complex. In thismodel, the doripenem molecule was built into the active site ofOXA-1 as an acyl-enzyme intermediate covalently attached tothe catalytic serine residue, Ser67. In preparation for modeling,the CR atom of Ser67 was truncated to a methyl group, creatingan ethyl ester linkage to the doripenem derivative. UsingHyperChem (21), hydrogens were added to create the appro-priate isomer and the structure was then geometry-optimizedusing PM3, in the gas phase, with a charge of -1.

FIGURE 1: (A) Structure of doripenem. (B) Conversion of the Δ2

doripenem tautomer to the Δ1 form after acylation.

1Abbreviations: SDS-PAGE, sodium dodecyl sulfate-poly-acrylamide gel electrophoresis; MPD, 2-methyl-2,4-pentanediol; PDB,Protein Data Bank; rmsd, root-mean-square deviation; KCX, carboxy-lysine residue.

11842 Biochemistry, Vol. 48, No. 50, 2009 Schneider et al.

RESULTS

In an effort to understand the inhibition of class D β-lactamases by carbapenems, the structure of the complex ofOXA-1 with doripenem was determined to 1.4 A resolution(Figure 2). A comparison of the complex with the apoenzymestructure shows strong similarity throughout the entire protein.Upon alignment of the two structures, an rmsd of 0.50 A wasdetermined for all CR atoms. Initial Fo - Fc electron densitymaps contoured at 3.0σ exhibited unambiguous density for thepresence of doripenem bound in each of the OXA-1 active sites.Continuous electron density connects the carbonyl carbon atom(C7) of doripenem and the Oγ atom of S67 (Figure 3A). Thisserine is known to act as the catalytic nucleophile (13), indicatinga covalent attachment between the drug and the enzyme.Additionally, clear density for all atoms of doripenem is ob-served, indicating that for the duration of the soak, no fragmen-tation of the drug occurs (Figure 3A). The final proteinmodel was analyzed with Procheck (22), showing that 95% ofthe non-proline, non-glycine residues are in the preferred regionof the Ramachandran plot (4% are in the additionallyallowed region).

The only significant effect of drug binding on the main chain isseen in an approximately 1 A movement of the loop formed byresidues 94-103, a shift that slightly closes the active site. Thisloop forms the upper rim of the active site and is drawn toward

the pyrrolidine/sulfonamide side chain of doripenem when thedrug is present.

It is noteworthy that K70 is clearly carboxylated in thepresence of doripenem (Figure 3A), and the resulting carbamateadopts essentially the same position observed in the apo struc-ture (14). The presence of this modification contrasts with theOXA-13-meropenem complex, in which case the authors con-cluded the analogous lysine (K70) is not carboxylated (5). Incontrast to the complexes of carbapenems with class A and classC β-lactamases, there is no water molecule within 5 A of thecarbamate of carboxy-K70 that could potentially act as thedeacylating water. Instead, the OQ1 atom of the carbamategroup makes a hydrogen bond to the hydroxyethyl group ofdoripenem [atom O62, 2.6 A (Figure 3B)]. This hydrogen bondlikely prevents the recruitment of the deacylating water, provid-ing a satisfying explanation for the inhibitory nature of doripe-nem and other carbapenems toward OXA-1.

Another strong contrast between our structure and those ofclass A and C enzymes is the position of the acyl carbonyl. In thecomplexes of imipenem with TEM-1 (3) and AmpC (2), andmeropenem with SHV-1 (8), this carbonyl is flipped 180� awayfrom the two main chain amide nitrogens that comprise theoxyanion hole (Figure 3C). The carbonyl group of doripenembound to OXA-1, on the other hand, forms two strong hydrogenbonds with the main chain amides of S67 (2.7 A) and A215

Table 1: Crystallographic Summary of the OXA-1-Doripenem Structure

cell constants a = 35.04 A, b = 71.61 A, c = 93.14 A, R = γ = 90�, β = 99.18�space group P21resolution (A) 1.40 (1.45-1.40)a

no. of unique reflections 86546

total no. of observations 440350

Rmerge (%) 7.4 (29.3)a

completeness (%)b 96.6 (88.1)a

ÆIæ/ÆσIæ 13.5 (4.2)a

resolution range for refinement (A) 50-1.4

no. of protein residues 498

no. of water molecules 434

rmsd for bond lengths (A) 0.013

rmsd for bond angles (deg) 1.36

R-factor (%) 18.3

Rfree (%)c 20.6

average B-factor (A2), protein atoms (molecule A) 14.4

average B-factor (A2), protein atoms (molecule B) 12.7

average B-factor (A2), doripenem atoms (molecule A) 22.3

average B-factor (A2), doripenem atoms (molecule B) 22.9

average B-factor (A2), water molecules 29.5

aValues in parentheses are for the highest-resolution shell. bFraction of theoretically possible reflections observed. cRfree was calculated with 5% of thereflections set aside randomly.

FIGURE 2: Stereoview of the overall structure of OXA-1 bound to doripenem. This and all subsequent figures were created with PyMOL (29).

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(2.8 A). This is consistent with the position of meropenem inOXA-13, thus establishing a pattern for the mode of carbapenembinding to class D β-lactamases.

A unique feature of the OXA-1-doripenem complex isthe interactions by which the carboxylate of the carbapenemis anchored to the enzyme. In most β-lactamase-β-lactam

complexes, a highly conserved arginine (e.g., R244 in TEM-1or R349 in AmpC) forms a salt bridge to the carboxylate, eitherdirectly or through a bridging water molecule (2, 3, 5, 8). For apoOXA-1, Sun et al. noted the lack of any arginine near this areaand suggested S258 may form a hydrogen bond with β-lactamsubstrates (14). Instead, the doripenem carboxylate forms a

FIGURE 3: (A) Stereoview of the 2Fo- Fc electron density maps contoured at 1.0σ surrounding doripenem and active site residues S67, KCX70,K212, and T213. Carbon atoms of OXA-1 are colored light blue, carbon atoms of doripenem green, nitrogens blue, oxygens red, and sulfursyellow. (B) Hydrogen bonding interactions (red dashes) between OXA-1 and doripenem. (C) Overlay of imipenem (salmon) in the active site ofTEM-1 (PDB entry 1BT5) (3) and doripenem (green) in OXA-1, showing the different orientations of the carbonyl oxygen with respect to theoxyanion hole (right side). Residue numbers are for OXA-1.

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hydrogen bond to the side chain of T213 and forms a salt bridgewith K212 (Figure 3B). There is an additional hydrogen bondfrom the carboxylate to one of two alternative conformations ofS115, a residue that has been proposed to shuttle a proton to thelactam nitrogen during catalysis (14). To contrast this novelmode of binding with the more common arginine interaction,we aligned the OXA-1-doripenem complex with the OXA-13-meropenem comoplex (5). The main chains align with anrmsd of 2.65 A (Figure 4A), with the entire ester linkage over-lapping quite well (Figure 4B). However, the meropenem anddoripenem structures greatly diverge starting with the pyrrolinering. Most notably, the interaction between the carboxylate andK212 causes a shift of the entire pyrroline ring back toward thehelix comprising residues 108-114 (helix A4). The trajectory ofthe pyrrolidine ring (i.e., the distal five-membered ring) andsulfonamide side chain differs greatly from that of the similar sidechain of meropenem, with the sulfonamide approaching the loopcomprising residues 97-99 (linking strands B5 and B6). Theamine of the pyrrolidine ring, which is likely protonated atpH 7.5, forms a hydrogen bond to the carbonyl of L255, whilethe sulfonamide interacts with the carbonyl oxygens of S237 andG238 of a symmetrically related molecule.

The side chain of V117 packs tightly against the hydroxyethylgroup of doripenem (Figure 5A), much as the homologousresidue of TEM-1 (N132) does with the same group of imipenem(Figure 5B). Both the β-branched alkyl side chain ofV117 and the

hydroxyethyl moiety of doripenem, however, show evidence ofmultiple rotational conformers, suggesting a degree of flexibilityin this interaction.

There has been much speculation about the role of carbape-nem tautomerization in themechanismof β-lactamase inhibition.While the positions of hydrogen atoms are not resolved in thisstructure, the resolution is sufficient to determine the geometryaround theC3 atom. In the intact drug, as well as theΔ2 tautomerproduced just after acylation, the pyrroline double bond causesC3 to be planar (Figure 1B). Isomerization to the Δ1 tautomerand the accompanying protonation of C3 creates an additionaltetrahedral center. The electron density of the sulfur attached toC3 clearly shows that that atom rises significantly above the planeof the pyrroline ring (Figure 6A). This suggests that doripenemdoes in fact tautomerize after acylation, forming the R config-uration of a novel chiral center at C3. To determine the idealgeometry around C3, we modeled an ethyl ester version of theacyl doripenem (no protein) using HyperChem (21). Aftersemiempirical geometry optimization using PM3, we found thatthe angle formed between the C3-S bond and the plane definedby atomsC2,C3, andC4was 50�. The same angle in our structurewas 31�, suggesting that interactions of the protein with thedoripenem side chain may prevent formation of the ideal tetra-hedral geometry at C3. The presence of an R chiral center at thisposition contrasts to the geometry observed for meropenembound to M. tuberculosis BlaC, in which the novel chiral center

FIGURE 4: Superposition of the structures of the OXA-13-meropenem complex (cyan) (5) with the OXA-1-doripenem complex (green). (A)Overall structural similarity of themain chains. (B)Comparisonof themodebywhichOXA-1 andOXA-13 stabilize the carbapenemcarboxylate.

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formed adopts the S configuration (9). Along with severalstructures modeled with an sp2 (planar) C3 atom (2, 3), theseresults highlight the diverse range of geometries that can be foundin acyl-carbapenem structures (Figure 6B).

DISCUSSION

The mode by which carbapenems inhibit β-lactamases is ofgreat interest; a thorough understanding of their mechanism ofaction may aid in the design of more effective inhibitors. More-over, understanding the lack of hydrolytic efficacy displayed bynarrow spectrum enzymes like OXA-1 toward carbapenemsshould help explain how that inhibitory nature is overcome bycarbapenemases. There are indeed many different possible ex-planations for the lack of carbapenem deacylation in class Dβ-lactamases, but analysis of the structure reported here pointsclearly in one direction.

When carbapenems acylate most class A and C β-lactamases,the ester carbonyl flips out of the oxyanion hole. This positivelycharged cavity is known to stabilize the tetrahedral transitionstate; thus, the conformational change provides a clear explana-tion for deacylation deficiency. This flip does not occur in theOXA-1-doripenem complex, showing that class D β-lactamasesare not inhibited by carbapenems in the same way as the othertwo classes. In TEM-1, this conformational change is caused by a

steric clash between the 6R-hydroxyethyl moiety and the sidechain of N132. It is likely that the somewhat smaller side chain ofV117 in the homologous position of OXA-1 accounts at leastpartly for this major structural difference. It is tempting tospeculate that substitution of asparagine (or an even largerresidue) for this valine might cause a rearrangement similar tothat seen for TEM-1, SHV-1, or AmpC.

Another potential inhibition mechanism that has been elimi-nated by this structure is deacylation deficiency caused bydecarboxylation of K70. We have recently shown that elimina-tion of carboxy-K70 in OXA-1 by mutagenesis leads to anenzyme that accumulates the acyl-enzyme intermediate ofpenicillin class β-lactams (13). Moreover, members of the BlaR1family of β-lactam sensor proteins are known to undergodecarboxylation of an active site carboxy-lysine after acylation,leading to the deacylation deficiency property that is necessaryfor their function (23-26). The fact that we see unambiguousdensity for the carboxyl moiety of K70 in our ligand complexshows that this mechanism is not applicable here. The lack oflysine carboxylation in the OXA-13-meropenem structure (5),as noted earlier, is most likely an artifact of crystallization pH.The lack of a hydrogen bond between the (missing) carboxy-lysine of OXA-13 and the hydroxyethyl moiety of meropenemmay contribute to the lateral shift of the ligand pyrroline ring(Figure 4B) compared to doripenem bound to OXA-1. The

FIGURE 5: Comparison of the carbapenem hydroxyethyl moiety for (A) doripenem and V117 of OXA-1 and (B) imipenem and N132 of TEM-1 (3).

11846 Biochemistry, Vol. 48, No. 50, 2009 Schneider et al.

presence ofR250 inOXA-13 and the lack of a similarly positionedarginine in OXA-1, however, also contribute to this lateral shift.Regardless of whether this shift reflects a true difference betweencarbapenems bound to different class D β-lactamases, the pre-sence of the carboxy-lysine in OXA-1 argues that the doripenemstructure we observe is the physiologically relevant form.

To complete its full catalytic cycle, OXA-1must recruit a watermolecule to the vicinity of carboxy-K70 after acylation. Thecarbamate, acting as a general base, then activates it for attack onthe ester bond. The fact that no water molecules are observednear the carbamate indicates that doripenem prevents recruit-ment of the deacylatingwater, providing a simple explanation fordeacylation deficiency and inhibition. The most likely sourceof interference in water recruitment is a hydrogen bond formedbetween the carbamate and the hydroxyethyl group of do-ripenem. In contrast, the hydroxyethyl group in bothTEM-1-imipenem and AmpC-imipenem complexes does notform a hydrogen bond to the putative general base (E166 inTEM-1 or Y150 in AmpC) but rather interacts with an aspar-agine (N132 in TEM-1 or N152 in AmpC). The general base inboth enzymes is therefore able to recruit a water molecule, albeitone that is unable to complete the deacylation event. Because theasparagine of TEM-1 is replaced with a nonpolar aliphaticresidue in all class D enzymes, the interaction between thecarbapenem hydroxyethyl group and the carboxy-lysine (andthus the mode of inhibition) is likely conserved throughout all ofthe class D enzymes.

The possibility of a tautomeric shift of the carbapenem aftercleavage of the lactam bond has been postulated from the earliest

description of interactions of carbapenem with β-lactamases(6, 15, 27, 28). Biphasic hydrolysis profiles gave rise to thehypothesis that the two tautomers are deacylated at differentrates. TheΔ2 form, which is produced directly from lactam bondcleavage of the parental compound, deacylates rapidly (in thismodel). Alternatively, tautomerization to the Δ1 pyrroline leadsto an acyl-penem conformation that is resistant to hydrolysis,causing inhibition. Isomerization of doripenem to the Δ1 tauto-mer is consistent with the spatial arrangement of the electrondensity around C3 in our structure, though alternate techniquesthat are sensitive to tautomeric differences (i.e., Raman spectro-scopy) will be needed for confirmation. One hypothesis suggeststhat tautomerization causes the carbapenem acyl carbonyl to flipout of the oxyanion hole in class A enzymes (6). Because weobserve doripenem’s acyl carbonyl in the oxyanion hole, thisclearly cannot be the cause of slow deacylation in the case ofOXA-1. In TEM-1, R244 has been suggested as a potentialgeneral acid for the protonation of imipenem C3 during thetautomerization (15). The side chain of doripenem is exposed tosolvent when bound to OXA-1, so the lack of a similarlypositioned arginine in this enzyme is not problematic fortautomerization.

While the presence of two hydrogen bonds between doripenemand a symmetry-related OXA-1 molecule may impact the con-formation of doripenem in the active site, we believe that thenovel set of interactions we observe are likely present in thenoncrystalline (i.e., solution) structure. The two crystal latticehydrogen bonds involve only the most distal portion of thedoripenem side chain (i.e., the sulfonamide group). The positionof the acyl carbonyl in the oxyanion hole and the position of thepyrroline ring and its carboxylate are stabilized by a large numberof hydrogen bonds and ionic interactions within the samemonomer unit. Also, the position of the pyrrolidine ring isstabilized by a hydrogen bond within the same protein unit(with the carbonyl of L255). Thus, the two groups that mostlikely impact the geometry of C3 and its attached sulfur atom(i.e., the pyrroline and pyrrolidine rings) are anchored by polargroups within the same protein unit. Attempts to model the Δ2

tautomer (instead of the Δ1 form that we observe) result in theloss of the hydrogen bond with L255 and also in steric clashesbetween doripenem and the side-chains of M99 and A215.

In conclusion, the OXA-1-doripenem structure revealsmechanisms of inhibition, carboxylate stabilization, and tauto-merization that differ from those observed in other β-lacta-mase-carbapenem complexes. This diversity reveals that subtlechanges in active site architecture, even among highly similarβ-lactamases, can result inmajor differences in ligand binding.Asmore structures become available in the future, these findings willcontribute to the understanding of why some β-lactamases areinhibited by carbapenems while others are able to hydro-lyze them.

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