Active-Site Plasticity Is Essential to Carbapenem Hydrolysis byOXA-58 Class D �-Lactamase of Acinetobacter baumannii

Shivendra Pratap,a Madhusudhanarao Katiki,a Preet Gill,b Pravindra Kumar,a Dasantila Golemi-Kotrab,c

Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand, Indiaa; Departments of Biologyb and Chemistry,c York University, Toronto,Canada

Carbapenem-hydrolyzing class D �-lactamases (CHDLs) are a subgroup of class D �-lactamases, which are enzymes that hydro-lyze �-lactams. They have attracted interest due to the emergence of multidrug-resistant Acinetobacter baumannii, which is notresponsive to treatment with carbapenems, the usual antibiotics of choice for this bacterium. Unlike other class D �-lactamases,these enzymes efficiently hydrolyze carbapenem antibiotics. To explore the structural requirements for the catalysis of carbapen-ems by these enzymes, we determined the crystal structure of the OXA-58 CHDL of A. baumannii following acylation of its ac-tive-site serine by a 6�-hydroxymethyl penicillin derivative that is a structural mimetic for a carbapenem. In addition, severalpoint mutation variants of the active site of OXA-58, as identified by the crystal structure analysis, were characterized kinetically.These combined studies confirm the mechanistic relevance of a hydrophobic bridge formed over the active site. This structuralfeature is suggested to stabilize the hydrolysis-productive acyl-enzyme species formed from the carbapenem substrates of thisenzyme. Furthermore, our structural studies provide strong evidence that the hydroxyethyl group of carbapenems samples dif-ferent orientations in the active sites of CHDLs, and the optimum orientation for catalysis depends on the topology of the activesite allowing proper closure of the active site. We propose that CHDLs use the plasticity of the active site to drive the mechanismof carbapenem hydrolysis toward efficiency.

Carbapenems, such as imipenem and meropenem, are �-lac-tam antibiotics with a wide spectrum of activity. Their clinical

introduction was driven by the need to overcome �-lactam resis-tance as a result of their efficacy in inhibiting most �-lactamases.However, as a result of selective pressure, �-lactamases acquiredthe ability to recognize and hydrolytically degrade the carbapen-ems. �-Lactamases hydrolyze the �-lactam bond of these antibi-otics. Four subclasses of the �-lactamases are identified, based ontheir amino acid sequences: A, B, C, and D. Classes A, C, and D are�-lactamases with a serine active site, and class B �-lactamases aremetalloenzymes. Importantly, the progressively increasing abilityof all four classes of �-lactamases to hydrolyze carbapenems hasbeen documented (1, 2).

Class D �-lactamases are also known as oxacillinases (OXA�-lactamases) due to their ability to hydrolyze the oxacillin sub-class of the penicillins (3). The OXA �-lactamases were firstobserved to be a resistance mechanism against imipenem in themid-1980s, coincidentally in the same year imipenem wasintroduced for clinical use (4, 5). Carbapenem-hydrolyzingclass D �-lactamases (CHDLs) are directly associated withoutbreaks of carbapenem-resistant Acinetobacter baumanniiaround the world. CHDLs have emerged as a major problem inthe treatment of multidrug-resistant A. baumannii, for whichcarbapenems were previously the drugs of choice (6–8). CHDLsnow are commonly found in all Acinetobacter species, thus in-creasing the risk of their being spread to other organisms. In-deed, CHDLs are also found in the Enterobacteriaceae, Klebsiellapneumoniae, and Pseudomonas aeruginosa (9). OXA-23 was thefirst CHDL to be identified (10) and, to date, there are close to 200variants of class D �-lactamases reported to possess carbapen-emase activity (9). Their genes are either plasmid borne or con-tained on chromosomes, and they are believed to be either re-cently acquired or native to A. baumannii, respectively. Sequencealignments indicate that CHDLs share 18% sequence identity with

the class D oxacillinases and 40 to 90% sequence identity with eachother (9).

CHDLs are divided into 12 distinct subgroups. The mostwidely spread carbapenemases in A. baumannii are OXA-23-like, OXA-40-like (prior name for OXA-24), OXA-51-like, andOXA-58-like �-lactamases (9). Another important CHDL isOXA-48. This enzyme is nonnative to A. baumannii and wasisolated from K. pneumoniae in 2001 (11). Since then, OXA-48and its variants have also been isolated in A. baumannii andother Enterobacteriaceae (12). Of all the CHDLs, only the struc-tures of OXA-24, OXA-23, OXA-146 (OXA-23 like), andOXA-48 have been solved, either alone or in complex withanother carbapenem (13–17). Additionally, the structure ofOXA-58 alone (referred to as apo-OXA-58) was recently re-ported (14). Structural analyses of these CHDLs show that theyshare key structural features with the class D �-lactamases,such as OXA-1 and OXA-10, including the carboxylated stateof the conserved catalytic residue Lys-86 (OXA-58 number-ing), which was first identified as being essential to the class D�-lactamase catalytic mechanism in class D �-lactamases (18).

Received 14 June 2015 Returned for modification 6 August 2015Accepted 3 October 2015

Accepted manuscript posted online 12 October 2015

Citation Pratap S, Katiki M, Gill P, Kumar P, Golemi-Kotra D. 2016. Active-siteplasticity is essential to carbapenem hydrolysis by OXA-58 class D �-lactamase ofAcinetobacter baumannii. Antimicrob Agents Chemother 60:75–86.doi:10.1128/AAC.01393-15.

Address correspondence to Pravindra Kumar, pravshai@gmail.com, orDasantila Golemi-Kotra, dgkotra@yorku.ca.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AAC.01393-15.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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Structural analyses and mutagenesis studies of CHDLs suggestthat two structural elements in CHDLs are responsible for the gainin carbapenemase activity by these enzymes: a shorter connectingloop of the �6- and �7-strands that form one side of the active site(19), and a hydrophobic bridge over the active-site cleft (13, 15,17). The second structural feature was observed in the crystalstructures of OXA-23 and OXA-24, and its presence was predictedin the homology model of the OXA-58 structure (20). It was notobserved in the crystal structure of OXA-58 solved by Smith et al.(14) or in the structure of OXA-48 (16). Therefore, OXA-58 andOXA-48 were proposed to have different hydrolytic mechanismstoward carbapenems (14).

In this study, we evaluated the structural elements that contrib-ute to the catalysis of carbapenems by OXA-58 by solving thestructure of this enzyme in an acyl-enzyme complex with the 6�-hydroxymethyl penicillin derivative (6�HMP) as a carbapenemstructural mimetic (Fig. 1). Analysis of the OXA-58 – 6�HMPcomplex structure revealed that the structural changes that takeplace on the two sides of the active-site cleft lead to the formationof a hydrophobic bridge over the OXA-58 active site. Two rotam-ers of the acyl-enzyme species are thus formed. We also carried outkinetic studies on several OXA-58 variants to probe the signifi-cance of the residues homologous to those that are involved in theformation of the hydrophobic bridge in OXA-23 and OXA-24.These findings shed new light on the relationship between theplasticity of OXA-58 in particular, and CHDLs in general, withrespect to the optimum binding mode to allow the hydrolysis ofcarbapenems.

MATERIALS AND METHODSChemicals and reagents. All reagents were American Chemical Society(ACS) grade and were purchased from either Merck Millipore, Sigma,Fluka, or Bio-Rad. Growth media were purchased from HiMedia Labora-tories (Mumbai, India) or VWR (Canada). Chromatography media andcolumns were purchased from GE Healthcare (Canada). Crystallizationscreens (Crystal Screen I & II, PEG/Ion I & II, Index, Salt, and CrystalScreen Cryo) were procured from Hampton Research (USA).

Protein crystallization. OXA-58 was produced and purified as de-scribed earlier (20). Purified OXA-58 was concentrated to 20 mg/ml in 10mM sodium phosphate (pH 7.0) buffer and 100 mM NaCl. Crystals ofOXA-58 were obtained using the vapor diffusion method in 96-well sit-ting drop plates (Hampton Research, Inc., Aliso Viejo, CA) at 293 K. Forthe initial crystallization screening, small drops were prepared by mixing1 �l of protein solution with the same volume of well solution and equil-ibrated against 50 �l of well solution. All of the listed crystallization kitswere evaluated. We obtained diffraction quality crystals of OXA-58 usinga well solution containing 0.1 M Tris-HCl (pH 8.0) buffer and 28% (wt/vol) polyethylene glycol 3350 (PEG 3350).

To obtain crystals of OXA-58 in the acyl-enzyme state, we soakedcrystals of native protein in a solution of 10 mM 6�-hydroxymethyl pen-icillin (6�HMP) or 6�-hydroxyoctyl penicillin (6�HOP) in 0.1 M Tris-HCl (pH 8.0) buffer and 30% (wt/vol) PEG 3350 for 15 min at roomtemperature.

Data collection and processing. The diffraction data sets for nativeand �-lactam-bound OXA-58 crystals were collected on an in-house X raysetup with a MAR 345 imaging plate detector mounted on a Bruke NoniusMicrostar-H rotating anode generator. For all crystal structures, the dif-fraction data were processed and scaled using HKL-2000 (21). The nativeprotein crystal belonged to the P212121 space group with the unit cell

FIG 1 Chemical structures of 6�HMP, 6�HOP, and imipenem (representative of carbapenems) and its acylated species with a �-lactamase.

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parameters a � 37.07, b � 67.03, and c � 93.51. The 6�HMP-boundOXA-58 crystals belonged to the P21 space group with the unit cell pa-rameters a � 36.94, b � 65.65, and c � 191.59, and � � 91.7°. The datacollection statistics are given in Table 1.

Structure modeling and refinement. The initial phases for OXA-58were obtained by molecular replacement with MolRep in the CCP4 suite(22, 23) using a monomer of the carbapenemase OXA-24 structure (Pro-tein Data Bank [PDB] code 2JC7, chain A) as a search model (24). Therigid body refinement was followed by iterative cycles of restrained coor-dinate and atomic displacement parameter refinement, including trans-lation-libration-screwmotion (TLS) refinement with Refmac5 (22) andPhenix.Refine (25). The repetitive cycles of manual model rebuildingbased on �A-weighted 2Fo�Fc and Fo�Fc maps were performed usingCOOT (26). The Fo�Fc difference map for the 6�HMP-bound form ofOXA-58 clearly showed density for 6�HMP above the 3� level, allowingthe 6�HMP structure to be modeled into the density. Water moleculeswere added in peaks that were simultaneously �3� in the Fo�Fc differ-ence map and �1� in the 2Fo�2Fc map. Several rounds of refinement andmodel rebuilding were performed until the best possible model wasachieved.

Programs used. The stereochemical attributes of the refined modelwere validated using the program MolProbity (28). The refinement sta-

tistics are presented in Table 1. All protein structure figures were preparedwith University of California, San Francisco (UCSF) Chimera (Resourcefor Biocomputing, Visualization, and Informatics, UCSF) (29). DynDomwas used to determine the hinge regions and calculate the rigid bodydomain movements involved in the conformational changes between apoand 6�HMP-bound OXA-58 (30).

Enzyme kinetics. OXA-58 F114A, F113A, M225T, M225A, F113Y,and F114I clones were generated by Mutagenex, Inc. (Hillsborough, NJ).OXA-58 and its variants were produced and purified as described earlier (20).The steady-state kinetics parameters kcat and Km for all of the mutant en-zymes, with imipenem and penicillins as substrates, were determined usingisothermal titration calorimetry (ITC) analysis in a MicroCal iTC-200 instru-ment (GE Healthcare), in accordance with the protocol first developed byPoduch and colleagues (31) and adopted for OXA-58 by Verma et al. (20).

Protein accession numbers. The atomic coordinates and the struc-ture factors for OXA-58 with and without ligand bound were deposited inthe Protein Data Bank (PDB) (27) with the accession numbers 4Y0O,4Y0T, and 4Y0U.

RESULTSDetermination of OXA-58 crystal structure. The 6-hydroxyalkylpenicillin derivatives have been described as good probes of the

TABLE 1 Data collection and refinement statistics

Data collection statistics

Value(s) for:

OXA-58 (apo structure) OXA-58–6�HMP complex OXA-58 (pseudoapo structure)

Wavelength (Å) 1.54 1.54 1.54Resolution range (Å) 33.52–2.37 (2.46–2.37) 36.93–2.60 (2.69–2.60)a 34.79–2.29 (2.37–2.29)Space group P212121 P21 P21

Unit cell a, b, c (Å); � (°) 37.07, 67.03, 93.51 36.94, 65.65, 191.59; 91.7 37.02, 65.10, 191.96; 91.28No. of total reflections 29,760 (1,605) 70,515 (4,578) 100,971 (7,493)No. of unique reflections 9,500 (721) 21,116 (1,637) 37,470 (2,741)Multiplicitya 3.1 (2.2) 3.3 (2.8) 2.8 (2.7)Completeness (%) 95.7 (73.1) 87.3 (69.3) 91.23 (74.8)Avg I/sigma(I) 11.26 (2.75) 8.45 (2.30) 9.15 (2.65)Wilson B-factor 34.76 39.21 33.11Rmerge 0.11 (0.36) 0.15 (0.43) 0.12 (0.42)Rmeas 0.13 0.15 0.14CC1/2 0.99 (0.77) 0.97 (0.68) 0.99 (0.78)CCa 0.99 (0.93) 0.99 (0.90) 0.99 (0.94)Rwork 0.19 (0.30) 0.21 (0.31) 0.19 (0.30)R-free 0.24 (0.36) 0.26 (0.36) 0.24 (0.36)

No. of nonhydrogen atoms 2,060 8,126 8,146Macromolecules 1,943 7,784 7,758Ligands 60Water 117 282 388

No. of protein residues 243 975 968RMS (bonds) (Å) 0.014 0.019 0.016RMS (angles) (°) 1.74 1.80 1.65

Ramachandran plot (%)Favored regions 98 96 99Outlier regions 0 0.41 0

Clash score 0.77 0.38 1.29

Avg B-factor 44.0 54.2 44.5Macromolecules 44.2 54.1 44.7Ligands 56.2Solvent 39.9 55.7 44.5

a Statistics for the highest-resolution shell are shown in parentheses.

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deacylation mechanism of serine �-lactamases (32–34). The ste-reochemistry at the C-6 position may direct the hydroxyalkylgroup of the penicillin to either the �-face or �-face of the �-lac-tam. Depending which face of the carbonyl of the acyl-enzymespecies is approached by the deacylating water molecule, one ste-reoisomer of the 6�- and 6�-hydroxyalkyl penicillin derivativeswill be a substrate, and the other stereoisomer will be an inhibitor.In the case of OXA-58, Verma et al. (20) showed that 6�HMP wasa substrate for OXA-58, while the 6�HMP stereoisomer behavedas a competitive inhibitor. The 6�HOP behaved as an inactivatorof OXA-58 (19). Carbapenems are 6�-hydroxyalkyl �-lactam de-rivatives. Hence, we set to understand the binding modes of 6�-hydroxyalkyl penicillin derivatives in order to understand thestructural elements required for the hydrolysis of carbapenems byCHDLs in general and OXA-58 in particular.

We attempted to solve the crystal structure of OXA-58 boundto 6�HOP (Fig. 1). The diffraction profile of the OXA-58 crystalafter being soaked in a 6�HOP solution for 15 min revealed thespace group of the crystal to be P21. The crystal diffracted to 2.29 Åresolution, and the OXA-58 structure obtained from this crystalrevealed four protein molecules per asymmetric unit. However,no ligand, either covalent or noncovalent, was found in the activesite. The final refined structural model of OXA-58 contained 968amino acid residues, 388 water molecules, and eight bicarbonatemolecules.

Soaking the OXA-58 crystals with 6�HMP for 15 min resultedin the formation of a stable acyl-enzyme species. The crystal of theOXA-58 – 6�HMP complex diffracted to 2.60 Å resolution andshowed the same space group (P21) and thus four molecules perasymmetric unit, as observed for the OXA-58 crystal soaked with6�HOP. The refined structural model of the OXA-58 – 6�HMPcomplex contained 975 amino acid residues, 282 water molecules,four 6�HMP molecules, and eight bicarbonate molecules.

Overall OXA-58 structure in unbound and bound states.Brief soaking of the OXA-58 crystal with 6�HOP led to a reduc-tion in the symmetry of the space group of the crystal, fromP212121 obtained for the OXA-58 alone to P21. An identical resultwas observed following soaking of the OXA-58 crystal with6�HMP, but in this case, a stable acyl-enzyme species was formed.We know that both penicillin derivatives interact with OXA-58,albeit with different dissociation constants, with a Ki of 83 3 �M(mean standard deviation) for 6�HMP and a Ki of 404 10�M for 6�HOP (20). Thus, the decrease in the space group sym-metry of the OXA-58 crystal soaked in the solution of either com-pound must be presumed to result from their binding to the en-zyme (whether covalently or noncovalently), effecting a structuralchange to the protein. An example of ligand binding reducing thespace group symmetry of the protein crystal, which is linked tothe induction of conformational changes in the protein struc-ture, is found in the literature: the flavin adenine dinucleotide(FAD)-linked quinone reductase 2 enzyme gave a reduction in thespace group symmetry and structural change upon the binding ofchloroquinone (35).

Here, we refer to the native OXA-58 crystal structure as apo-OXA-58. Our apo-OXA-58 and that of Smith et al. (14) superim-pose with a backbone root mean square deviation (RMSD) of 0.31Å among 243 atom pairs (Fig. 2). The crystal structure of OXA-58solved from soaking with 6�HOP is referred to as pseudoapo-OXA-58. The apo and pseudoapo structures superimpose, with anRMSD of 0.36 Å among 242 atom pairs.

The noncovalent binding of 6�HOP to OXA-58 caused twonoticeable changes in structure, localized in the loops that connect�3- and �4-helices and �6- and �7-strands (Fig. 2). The largesteffect on these loops was noted for chain B. The �3/�4-loop har-bors residues Phe-113 and Phe-114, and the �6/�7-loop harborsMet-225. The homologues of Phe-114 in OXA-23 (Phe-110) andOXA-24 (Tyr-112) form a hydrophobic bridge with a methionineresidue (Met-221 in OXA-23 and Met-223 in OXA-24) located inthe �6/�7-loop (15, 17). In OXA-48, the homologues of Phe-114and Met-225 (Ile-102 and Thr-244, respectively) do not engage inhydrophobic interactions (16). Phe-113 of OXA-58 has no homo-logue in OXA-23, OXA-24, or OXA48; instead, a polar residue isfound at this position in both enzymes (Ser-109, Thr-111, andAsp-101, respectively).

The conformational change in the �3/�4-loop of pseudoapo-OXA-58 was associated with an inward movement by as much as1.79 Å (measured for chain B) in comparison to the apo-OXA-58structure (Fig. 2B). The inward movement in the �3/�4-loop wasalso associated with the rearrangement of the side chains of Phe-113 and Phe-114 and shifts by 0.97 Å and 0.86 Å of their respectiveC� atoms, in comparison to the apo structure (Fig. 2B). The tran-sient binding of 6�HOP also led to an overall 0.66-Å inwardmovement of the �6/�7-loop (homologues of �4/�5 in OXA-24/OXA-23 and �5/�6 in OXA-48) (Fig. 2C). The conformationalchanges in the �3/�4- and �6/�7-loops led to an overall 2-Å de-crease in the distance between the C� atoms of Phe-113/Met-225and Phe-114/Met-225, from 14.45 Å and 15.56 Å in the apo struc-ture to 12.76 Å and 14.18 Å in the pseudoapo structure. Thisinward movement exemplifies the key structural plasticity in theactive site of OXA-58.

The above-mentioned conformational changes in the �3/�4-and �6/�7-loops were more pronounced in the OXA-58–6�HMPcomplex, especially in the �6/�7-loop (Fig. 3). We measured a1.03-Å inward shift of the �6/�7-loop (calculations made forchain A). Notably, this movement was associated with a signifi-cant reorientation of the Met-225 side chain. In the OXA-58 –6�HMP complex, Met-225 is pointed toward Phe-113 and notPhe-114, as seen in the apo-OXA-58, OXA-23–meropenem, andOXA-24 – doripenem complexes (Fig. 3). The movements of bothloops brought the side chains of Phe113/Phe114 and Met-225closer: the distances between the C� atoms of Phe-113/Met-225and Phe-114/Met-225 changed from 14.45 Å and 15.56 Å in theapo complex to 12.06 Å and 13.67 Å, respectively, in the boundcomplex. In OXA-23, the distance between the C� atoms of Phe-110 and Met-221 is 13.89 Å, and in the OXA-24 structure, thecorresponding distance between Tyr-112 and Met-223 is 14.27 Å.The homolog residues of Phe-114 and Met-225 engage in hydro-phobic interactions in OXA-23 and OXA-24, leading to the for-mation of the tunnel-like topology of their active sites (15, 17). Inthe case of the OXA-58 – 6�HMP complex, the side chains of theabove-mentioned residues come also within the limits of van derWaals interactions (4.09 Å for Phe-113/Met-225 and 5.30 Å forPhe-114/Met-225 [distances measured between the C- of thephenyl ring and the sulfur atom of methionine]).

Analysis of the apo and 6�HMP-bound OXA-58 structures(chain A) by the DynDom server (30) identified two rigid domainsin the protein. The first encompassed 214 residues from 42 to 103to 124 to 276, and the second domain consisted of 20 residuesfrom 104 to 123. The two rigid regions were connected through ahinge composed of the flexible residues 103 and 104 (. . .EI. . .)

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and 122 and 123 (. . .TL. . .) (see Fig. S1 in the supplemental ma-terial). The binding of 6�HMP to OXA-58 induced an interdo-main closure motion of 8.3° around a rotation axis close to theflexible residues. This finding further exemplifies the plasticity ofthe OXA-58 active site and draws our focus to the domain closurethat occurs upon covalent binding of the �-lactam.

Active site of OXA-58 in the native and bound states. �-Lac-tam binding, either noncovalent or covalent, did not alter sig-nificantly the conformation of the key catalytic residues in theactive site (Fig. 3). In the pseudoapo and 6�HMP-boundOXA-58 structures, we observed that the carboxyl group of thecarboxylated Lys-86 was stabilized by hydrogen bonding toTrp-169 (3.04 Å), the O-� of Ser-83 (2.57 Å), and a watermolecule (Fig. 3). This network is similar to the hydrogen bondnetwork seen in the OXA-58 structure (the apo-OXA-58 struc-ture in this study or the structure solved by Smith et al. [14]). In

both apo/pseudoapo structures, the O-� of Ser-83 formed ahydrogen bond with the O-� of Ser-130 of the SNV motif, withthe O-� of Ser-130 also hydrogen bonding with N- of Lys-220of the K(S/T)G motif. Formation of the acyl-enzyme speciesbrought about rotation of the O-� atom of Ser-130 by 76°toward the N atom of 6�HMP.

The apo-OXA-58 structure contains two water molecules (W1and W2) in the active site, as was also observed in the Smith et al.(14) structure of OXA-58 (Fig. 3). W1 is positioned in the oxyan-ion hole by hydrogen bonding to the backbone amide protons ofTrp-223 and Ser-83. W2 bridges the carboxylated Lys-86 and Ser-83. A third water molecule, W3, which hydrogen bonds to Trp-169 and carboxylated Lys-86, was seen in the apo, pseudoapo, and6�HMP-bound structures. The transient binding of 6�HOP ledto the displacement of W2 (it is not observed in the pseudoapostructure). Formation of the acyl-enzyme species led to the dis-

FIG 2 Superposition of crystal structures of apo-OXA-58 (PDB code 4Y0O)and pseudoapo-OXA-58 (PDB code 4Y0T) and the native crystal structure ofOXA-58 reported by Smith et al. (14) (PDB code 4OH0). (A) Ribbon representation of apo (blue), pseudoapo (green), and 4OH0 (orange) structures of OXA-58.The �3/�4- and �6/�7-loops are indicated by an arrow. (B) Close-up view of the �3/�4-loop and residues Phe-113 and Phe-114 in apo (blue) and pseudoapo(green) structures. (C) Close-up view of the �6/�7-loop in apo (blue) and pseudoapo (green) structures.

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placement of both W1 and W2, but not W3, from the active site(Fig. 3).

Binding mode of 6�HMP to the active site of OXA-58. Nota-bly, we observed that the hydroxymethyl group of 6�HMP occu-pies two different conformations in the active site of OXA-58 (seeFig. S2 in the supplemental material). This group occupies thesame position in chains A, C, and D (Fig. 4A). In contrast, in chainB, the hydroxyl group is rotated by 60° (counterclockwise) fromthe first position, toward the back of the active site (Fig. 4B and 5).We compared the OXA-58 6�HMP structure (chains A and B)with the structures of the deacylation-deficient OXA-24 variant(Lys84Asp) bound to doripenem (PDB code 3PAE) and to that ofthe deacylation-deficient wild-type OXA-23 bound to mero-penem (PDB code 4JF4 [Fig. 5]). The wild-type OXA-23 was ren-dered deacylation deficient by crystallization at low pH (13). Su-perposition of the OXA-23–meropenem, OXA-24 – doripenem,and OXA-58 – 6�HMP structures shows that the hydroxymethylgroup of each carbapenem side chain is about 65° (clockwise)away from the position of the 6�HMP hydroxymethyl group seenin chains A, C, and D and closer to the entrance of the active site(Fig. 5). Each carbapenem molecule bound to OXA-23 or OXA-24adopts the �2-pyrroline tautomer (Fig. 1) in the respective crystalstructure of its acyl-enzyme species. These acyl-enzyme speciesrepresent the hydrolysis-productive species that OXA-24 andOXA-23 form with doripenem and meropenem, respectively,during catalysis. The fact that the hydroxyl group in 6�HMP ispositioned away from this orientation, toward the back of theactive site, may explain the stability of this acyl-enzyme species of6�HMP. At this conformation, the hydroxyl group of 6�HMPforms a hydrogen bond with the carboxylated Lys-86 (Fig. 5),which may dampen the basicity of carboxylated Lys-86 toward thedeacylating water molecule. Moreover, the conformation of6�HMP in chain B may also provide a physical barrier to theapproach of the deacylating water molecule. Incidentally, the6�HMP rotamer seen in chains A, C, and D of OXA-58 is similarto that seen in 6�HMP bound to TEM-1 (PDB code 1TEM) (seeFig. S3 in the supplemental material) (33). In the TEM-1– 6�HMPcomplex, it was noted that the hydroxyl group of 6�HMP dis-placed the structurally conserved deacylating water molecule, and

that hydrogen bonding with Glu-166 may prevent the glutamatefrom activating an incoming water molecule.

Both conformations of the 6�HMP side chain in OXA-58 arestabilized through hydrophobic interactions between the 6�HMPside-chain methylene carbon and the side chains of the conservedresidues Val-132 (4.18 Å apart) and Leu-170 (2.67 Å apart) (Fig.5). Of note, these two residues are in closer proximity to the6�HMP side-chain methylene carbon in OXA-58, in comparisonto their homolog residues in OXA-23 (13), OXA-24 (36), andOXA-48 (37) (Fig. 5; see also Fig. S4 in the supplemental material).The apparent movement of the -loop so as to be positionedcloser to the 6�HMP side chain may be due to an intrinsic abilityof the -loop, where Leu-170 is located, to interact with the alkylgroup of the carbapenem side chain so as to enable efficient catal-ysis (see below).

As a result of the inward movement of the �3/�4-loop in OXA-58 – 6�HMP, Phe-114 is within hydrophobic interaction contactwith the methyl groups at the C-3 position of 6�HMP (4.37 Å).These methyl groups are also in van der Waals contact with theIle-260 side chain (3.92 Å), located at the very back of the activesite. In addition, Trp-117 located in the �3/�4-loop is in van derWaals contact (4.14 Å) with the plane of the thiazolidine ring of6�HMP. Overall, these interactions create a sandwich effect withrespect to the �-lactam (Fig. 6). The sandwich effect, which acy-lated carbapenem molecules are also likely to experience, togetherwith the hydrophobic bridge formed over the active site, may ef-fect preferential stabilization of the �2-tautomer of carbapenems(Fig. 1). Stabilization of the �2-tautomer of the carbapenem willput the side chain at the C-3 carbon of carbapenem away from theside chains of Phe-114 and Phe-113 and avoid steric hindranceamong them; as a result, this will lead to more stable acyl-enzymespecies.

Kinetic characterization of wild-type and OXA-58 variants.We substituted other amino acids for the Phe-113, Phe-114, andMet-225 residues (Table 2) in order to explore their effects oncatalysis. Homologues of Phe-114 and Met-225 in OXA-23 andOXA-24 contribute to the tunnel-like topology of the active site inthe native structure of these enzymes. Phe-113, a position that isoccupied by Ser-109 and Thr-111, respectively, in OXA-23 and

FIG 3 Stereo diagram of superposition of crystal structures of apo (blue), pseudoapo (green), and OXA-58 – 6�HMP complex (chain A, in red; PDB code 4Y0U).The depicted side chains and 6�HMP are shown in a stick representation. The Fo-Fc omit electron density maps, contoured at the 3� level, indicating the positionand conformation of the 6�HMP in OXA-58 are displayed in green. Water molecules are shown as green spheres in the apo structure (W1, W2, and W3) and asorange spheres in the pseudoapo structure (W1= and W3=).

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OXA-24, seems to play a role in OXA-58 catalysis, as the Met-225is oriented toward Phe-113 in the acyl-enzyme species.

The steady-state kinetics for the wild-type and OXA-58 vari-ants were determined for imipenem and a number of penicillins.The results are summarized in Table 2. The mutation of Phe-113or Phe-114 to Ala had a negative impact, by as much as 5-fold, onthe efficiency of imipenem turnover (Table 2). This mutation af-fected both the binding affinity for imipenem (Km; 1.7-fold reduc-tion) and the turnover rate constant (kcat; 3-fold reduction). Ofthe penicillins assessed, the greatest impact observed for theF113A and F114A variants was on the turnover of oxacillin (Km

increases of 4- and 36-fold, respectively, and kcat decreases of 8-and 2.2-fold, respectively). The mutation of Phe to Ile at position114 had the most deleterious impact on the activity of OXA-58against imipenem, a 50-fold reduction mainly expressed on kcat. Asimilar effect on the kcat was also measured for the penicillins, with

a reduction in kcat of as much as 18-fold for penicillin G and37-fold for oxacillin (Table 2). The effect of the F114I substitutionon the activity of OXA-58 might result from steric clashes betweenthe isoleucine side chain and the Phe-113 side chain, with a loss ofthe optimal orientation for the interaction Phe-113 with Met-225.Moreover, the deleterious effects of this mutation point to thepotential role of Phe-114 in properly orienting the Phe-113 sidechain. This role is supported by the smaller effect measured for thePhe-to-Tyr mutation at position 113.

The effect of the Met-to-Thr substitution at position 225 on theactivity of OXA-58 mirrors that of the Phe-to-Ala substitution atposition 113. These observations suggest that threonine, with itsshorter and branched side chain and having a polar group at-tached to the C-�, may not allow interaction with Phe-113. Incontrast, the M225A mutation, involving a reduction in the side-chain mass, has a minor effect on enzyme activity. This outcome

FIG 4 Close-up view of the active-site structural elements in chain A (A) and chain B (B) of the OXA-58 – 6�HMP complex. The 6�HMP is shown in a stickrepresentation. The Fo-Fc omit electron density maps, contoured at the 3� level and indicating the position and conformation of the 6�HMP in chains A and Bof OXA-58 – 6�HMP, are displayed in green.

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might be due to the flexibility of the �3/�4-loop, which can movecloser to the �6/�7-loop to compensate for the loss in carbonchain length at this position.

DISCUSSION

The crystal structure of the OXA-58 – 6�HMP complex illumi-nates two important observations. First, the �3/�4- and �6/�7-loops move toward each other, leading to the formation of a hy-drophobic bridge across of the active site. Analysis of apo and6�HMP-bound OXA-58 structures using the DynDom server(30) revealed that binding of 6�HMP to OXA-58 induces an in-terdomain closure motion of 8.3°. The second important obser-vation is the ability of the hydroxymethyl group of 6�HMP tooccupy two positions in the active site of OXA-58. The more-populated rotamer (found in three out of four chains) is about 65°away from the rotamer of meropenem or doripenem observed instructures of the OXA-23–meropenem and OXA-24 – doripenemcomplexes (Fig. 5). The less-populated rotamer of 6�HMP is

about 120° away from these carbapenem rotamers (Fig. 5). Thesefindings directly impact our understanding of the structural fac-tors critical for carbapenem catalysis by class D �-lactamases.

The inward movements that the �3/�4 and �6/�7-loops expe-rience in the OXA-58 – 6�HMP complex lead to an �2-Å decreasein the distance between the C� atoms of the Phe-113/Phe-114 andMet-225. This decrease is significant, considering that the sidechains of these residues are brought into van der Waals contactwith each other. Any further decrease would lead to steric clashesamong these residues. Moreover, the conformational changes inthe �3/�4- and �6/�7-loops are also associated with rearrange-ment of the Phe-113/Phe-114 and Met-225 side chains. Notably,Met-225, which in the apo and pseudoapo structures of OXA-58 isoriented toward Phe-114 (as in the cases of OXA-23 and OXA-24), is repositioned so as to point toward Phe-113. As a result, theside chains of Phe-113/Phe-114 and Met-225 are at the properdistance and orientation to establish close hydrophobic interac-tions. This interaction coincides with formation of a hydrophobic

FIG 5 Superposition of active-site structural elements of chains A (red) and B (gray) of OXA-58 – 6�HMP complex, OXA-23–meropenem complex (PDB code4JF4, yellow), and OXA-24 – doripenem (PDB code 3PAG, magenta) complex. The ligands are shown in a stick representation and color-coded as the respectiveprotein ribbon color: 6�HMP bound in chain A is presented in red, 6�HMP bound in chain B is presented in gray, meropenem is shown in yellow, anddoripenem is shown in magenta. The arrow labeled “1” indicates the shift (60°) in position of the hydroxymethyl groups in 6�HMP bound to chain B of OXA-58with respect to the same group in chain A. The arrow labeled “2” indicates the shift in positions (65°) of the hydroxymethyl groups in meropenem (green) boundto OXA-24 and in doripenem (red) bound to OXA-23 with respect to the same group in chain A.

FIG 6 Molecular surface representation of the binding pocket of 6�HMP in the active site of OXA-58 – 6�HMP (chain A in red). The side chains of Trp-117,Phe-113/114, Met-225, and Ile260 are shown in balls and sticks. 6�HMP is represented as spheres and colored cyan (C atoms), red (oxygen atoms), and yellow(sulfur atoms). The snapshots are of same molecule rotated 180° horizontally.

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bridge above the active site, like a lid over the acyl-enzyme species,and it exemplifies the plasticity of the OXA-58 active site (Fig. 7).A side-by-side comparison of the molecular surfaces of the OXA-58 – 6�HMP, pseudoapo, and apo structures indicates that theactive-site closure is likely to be initiated upon Michaelis-Mentencomplex formation and is further established with the acyl-en-zyme species formation (Fig. 7).

The active-site plasticity of OXA-58 allows extensive hydro-phobic interactions between 6�HMP and the side chains of Phe-114, Trp-117, and Ile-260 during catalytic turnover. One mustinfer that �-lactams bind snugly to the active site of OXA-58, andthis snugness correlates (in the case of carbapenem substrates) topreferential stabilization of the �2-tautomer of the ring-openedcarbapenem (Fig. 1) and persistent occupation of the oxyanionhole by the carbonyl oxygen of the acyl-enzyme species. Thesefeatures play an important role in driving the reaction toward theformation of productive acyl-enzyme species (20, 36).

The above-mentioned hypothesis is well supported by the ef-fects that substitutions at Phe-113, Phe-114, and Met-225 had onthe activity of OXA-58. The replacement of Phe with Ile at posi-tion 114 had a substantial and deleterious effect on the hydrolysisof imipenem and penicillins, predominantly on the kcat values(50-fold decrease for imipenem). This substitution may affect theformation of the hydrophobic bridge through a lack of properorientation of Phe-113 and removal of the sandwiching effect thatthe �-lactam experiences while bound to the active site (seeabove). In all, the loss of the snugness in �-lactam binding may

lead to lower acylation rates and/or formation of nonproductiveacyl-enzyme species; both scenarios will result is smaller kcat val-ues. The formation of nonproductive acyl-enzyme species mayresult from a lack of locking of the carbonyl O atom of acyl-en-zyme species into the oxyanion hole, and/or hindrance of the car-bonyl carbon from the incoming deacylating water molecule.Both scenarios have been reported for the TEM-1–imipenemcomplex. In that case, the carbonyl oxygen of acyl-enzyme speciesdid not remain bound into the oxyanion hole, and the hydroxylmoiety of the imipenem side chain displaced the deacylating watermolecule (PDB code 1BT5) (38).

The replacement of Phe-113 or Phe-114 with Ala led to a de-crease in the catalytic efficiency of the enzyme for imipenem andpenicillins as a result of a negative effect on both the kcat and Km

values. However, the replacement of Phe with Tyr at position 113had very little effect on hydrolysis of all the tested �-lactams, in-dicating the importance of an aryl ring at this position to interactwith Met-225. In addition, the replacement of Met-225 with Alaalso had little effect on hydrolysis and might reflect the plasticity ofboth the loops, �3/�4 and �6/�7, in compensating for a shorterside chain at this position.

The hydrophobic bridge formed in the OXA-58 – 6�HMPstructure was also noted in the native structures of OXA-24 andOXA-23 (13, 15). This hydrophobic bridge remains intact in theirrespective structures as acyl-enzyme species (13, 36). The nativestructure of OXA-48 does not show this hydrophobic bridge (16).However, a recent structure of an OXA-48 –avibactam acyl-en-

TABLE 2 Steady-state kinetic parameters for OXA-58 and variantsa

Kinetic data by antibiotic WT M225A F113Y F114A F113A M225T F114I

Penicillin Gkcat/Km (�M�1 s�1) 15 3 19 4 20 2 7 1 4 1 4.1 0.5 0.58 0.06kcat (s�1) 106 10 141 29 137 9 124 8 37 7 42 3 5.7 0.5Km (�M) 7 1 7.3 0.7 6.9 0.7 17 2 10.3 2.9 10 1 9.7 0.2

Ampicillinkcat/Km (�M�1 s�1) 4.6 0.9 1.6 0.3 1.6 0.3 1.5 0.4 0.83 0.09 0.52 0.08 0.13 0.02kcat (s�1) 97 14 75 8 108 4 188 25 39 1 38 4 6.80 0.03Km (�M) 21 3 46 9 66 11 127 25 48 5 72 9 54 9

Carbenicillinkcat/Km (�M�1 s�1) 1.7 0.4 3.3 0.5 1.1 0.3 0.6 0.3 0.31 0.05 0.25 0.07 0.06 0.02kcat (s�1) 272 13 102 15 167 15 173 64 44 4 41 4 14.8 0.7Km (�M) 160 40 31 2 153 47 268 65 142 16 161 39 248 66

Amoxicillinkcat/Km (�M�1 s�1) 2.8 0.5 3.5 0.7 2.9 0.8 1.4 0.1 0.61 0.04 0.7 0.1 0.10 0.02kcat (s�1) 62 10 97 15 79 7 204 14 24 1 32 4 8.2 0.7Km (�M) 22 2 28 4 27 7 145 5 40 2 49 4 80 10

Oxacillinkcat/Km (�M�1 s�1) 3.5 0.4 3.2 0.4 1.1 0.3 0.05 0.02 0.11 0.02 0.036 0.005 0.0110 0.0002kcat (s�1) 137 14 179 14 85 6 59 19 17 3 17 2 3.69 0.04Km (�M) 39 3 56 4 74 21 1400 500 156 1 462 24 335 6

Imipenemkcat/Km (�M�1 s�1) 0.5 0.2 0.52 0.09 0.48 0.08 0.10 0.03 0.09 0.02 0.090 0.007 0.011 0.001kcat (s�1) 1.2 0.2 0.63 0.02 0.9 0.1 0.442 0.008 0.11 0.01 0.19 0.01 0.024 0.001Km (�M) 2.3 0.5 1.2 0.2 2.0 0.2 4 1 1.1 0.2 2.1 0.2 2.2 0.2

a Each progress curve was fitted to the nonlinear equation to obtain the kcat and Km. The means and standard deviations shown were calculated from the results from threeindividual experiments. The enzyme concentration varied from 5 nM for the wild-type (WT) enzyme to 200 nM for the F113L variant.

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zyme complex showed a partial closure of the active site as a resultof inward movement of the �3/�4-loop (37). Incomplete closureof the active site in OXA-48 is not an indication that OXA-48 is anoutlier among CHDLs using this structural feature for the catalysisof carbapenem hydrolysis, or that it brings no functional benefit tocatalysis by these enzymes. In fact, the native structure of OXA-48shows that the active site is more crowded with hydrophobic res-idues (16). In light of our findings, the crowdedness of the activesite may be just a different way to meet the requirement for atight-fit binding of carbapenem in the active site of this enzyme incomparison to OXA-58, OXA-23, and OXA-24, which may useadjustable active-site closure to meet this requirement.

Despite the tight fit of the 6�HMP acyl enzyme in the OXA-58active site, we observe that the hydroxymethyl group of 6�HMPoccupies two different conformations in the active site. Both con-formations of the 6�HMP side chain are stabilized through hy-drogen bonding between the hydroxyl group of 6�HMP and ei-ther Trp-223 (chains A, C, and D) or carboxylated Lys-86 (chainB) (Fig. 5) and hydrophobic interactions between the 6�HMPside-chain methylene carbon atom and the side chains of the con-served residues Val-132 (4.18 Å apart) and Leu-170 (2.67 Å apart).Docquier et al. (16) proposed that Val-132 and Leu-170 play a rolein the optimal positioning of the hydroxyethyl side chain of car-bapenems for catalysis by enabling the deacylating water moleculeto approach the carbon of the carbonyl of the acyl-enzyme species.It is peculiar that the -loop, which harbors Leu-170, is posi-tioned closer to the active site of OXA-58 than to OXA-23 andOXA-24. This structural feature seems to have an effect on thecontact that Leu-170 establishes with the methylene carbon atomof 6�HMP and might be a reflection of an intrinsic property of the -loop to interact with the side chains of substrates, with the

purpose of positioning them correctly for retaining the deacylat-ing water molecule.

A comparison of the two rotamers of a 6�HMP hydroxymethylside chain with the hydroxyethyl side chains of meropenem anddoripenem, seen in their respective complexes with OXA-23 (PDBcode 4JF4) (13) and OXA-24 (PDB code 3PAG) (36) (Fig. 5), showthat the rotamer of 6�HMP seen in chain B is further away fromthe entrance of the active site and is less populated; also, the rota-mer seen in the meropenem/doripenem bound to OXA-24 –OXA-23 complex is closer to the entrance of the active site and isthe only rotamer seen in these complexes. The rotamer of 6�HMPseen in chains A, C, and D occupies an intermediate conformationbetween the two rotamers mentioned above and has an occur-rence that is at a 3:1 ratio with the rotamer seen in chain B (Fig. 5).Looking at these three rotamers (two rotamers of 6�HMP and thevirtually identical rotamers of meropenem and doripenem), a se-quence of key events that may take place during carbapenem ca-talysis emerges. The first rotamer (chain B) may correspond to theacyl-enzyme complex formed from the Michaelis-Menten com-plex, while the second rotamer (chain A) may represent an inter-mediate conformation obtained while the hydroxyethyl side-chain samples the conformational space for optimal orientationfor catalysis in the active site of CHDLs. The third rotamer (seen inOXA-23–meropenem and OXA-24 – doripenem complexes) mayshow the optimal location of the hydroxyethyl side chain for thelast step of catalysis, deacylation. A deacylation step in �-lactamcatalysis by OXA �-lactamases requires that a water molecule be inhydrogen bond distance from the carboxylysine active-site residue(Lys-86) and the carbonyl moiety of the acyl-enzyme species. Theproposed flow of catalytic events suggests that class D �-lactama-ses may have acquired carbapenemase activity by selecting a rota-

FIG 7 Snapshots of active-site closure in OXA-58 induced by 6�HMP. The Phe-113, Phe-114, and Met-225 side chains implicated in the formation of thehydrophobic bridge over the active-site cleft are shown in orange, cyan, and magenta, respectively. Shown are surface diagrams depicting the active-site pocketof the apo structure (A), the pseudoapo structure (B), and the OXA-58 – 6�HMP complex (C). (D) Side-by-side comparison of the overall structures of apostructure (left) and OXA-58 – 6�HMP complex (right) depicted in molecular surface diagrams.

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mer conformation that allows not simply retention of the deacy-lating water molecule but its retention in the position between thecarboxylysine and the carbonyl of the acyl-enzyme complex, asrequired for catalytic deacylation.

Our study demonstrates that the active site of OXA-58 has theflexibility to mold around the ligand during catalysis. This featuremay be shared and optimized by other CHDLs to provide a tight-fitbinding for the carbapenem substrates. In addition, our study sug-gests that the OXA-58 active site enables the carbapenem substrates tosample and assume an optimum conformation for catalysis. Thesestructural features may enable the formation of a hydrolysis-produc-tive acyl-enzyme species, thereby identifying the essential catalyticdifference between the class D �-lactamases capable of carbapenemhydrolysis (the CHDLs) and the class D �-lactamases incapable ofcarbapenem hydrolysis. Last, it is plausible that in the presence ofantibiotic pressure, these structural elements are modulated toachieve catalytic efficiency in carbapenems.

ACKNOWLEDGMENTS

We thank Shahriar Mobashery (University of Notre Dame) for providingus with the 6�-hydroxymethyl- and 6�-hydroxyoctyl-penicillin deriva-tives. We thank Macromolecular Crystallographic Unit, a Central Facility(MCU) at IIC, IIT Roorkee, for purification, crystallization, data collec-tion, and structure determination. P.K. and S.P. thank ICMR, India, forproviding the financial assistance for these structural studies throughgrant 64/3/2012-BMS.

This work was in part supported by an Early Researcher Award toD.G.K. from the Ontario Ministry of Economic Development and Inno-vation, Ontario, Canada.

FUNDING INFORMATIONIndian Council of Medical Research (ICMR) provided funding to Shiven-dra Pratap and Pravindra Kumar under grant number 64/3/2012-BMS.Ontario Ministry of Economic Development and Innovation (Ministredu Développement Économique et de L’Innovation) provided funding toDasantila Golemi-Kotra under grant number ER09-06-134.

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