Structural insights into alterations in the substrate spectrum of serine-β-lactamase OXA-10 from Pseudomonas aeruginosa by single amino acid substitutions

ABSTRACT

The extensive use of β-lactam antibiotics has led to significant resistance, primarily due to hydrolysis by β-lactamases. OXA class D β-lactamases can hydrolyze a wide range of β-lactam antibiotics, rendering many treatments ineffective. We investigated the effects of single amino acid substitutions in OXA-10 on its substrate spectrum. Broad-spectrum variants with point mutations were searched and biochemically verified. Three key residues, G157D, A124T, and N73S, were confirmed in the variants, and their crystal structures were determined. Based on an enzyme kinetics study, the hydrolytic activity against broad-spectrum cephalosporins, particularly ceftazidime, was significantly enhanced by the G157D mutation in loop 2. The A124T or N73S mutation close to loop 2 also resulted in higher ceftazidime activity. All structures of variants with point mutations in loop 2 or nearby exhibited increased loop 2 flexibility, which facilitated the binding of ceftazidime. These results highlight the effect of a single amino acid substitution in OXA-10 on broad-spectrum drug resistance. Structure–activity relationship studies will help us understand the drug resistance spectrum of β-lactamases, enhance the effectiveness of existing β-lactam antibiotics, and develop new drugs.

GRAPHICAL ABSTRACT

Introduction

The discovery of penicillin in 1929 and its successful clinical use in the 1940s revolutionized the treatment of bacterial infections. For more than 70 years, different antibiotics have led to remarkable advances in the field of antimicrobial chemotherapy [1]. However, the overuse and misuse of antibiotics have led to the emergence and dissemination of resistance. Antibiotic resistance can be caused by several mechanisms, including target modification, downregulation of porins for antibiotic entry, overexpression of drug efflux systems, and production of antibiotic-modifying or degradative enzymes [2]. Therefore, antibiotic resistance is a remarkable threat to humankind.

The β-lactam antibiotics, such as penicillins, carbapenems, cephalosporins, and monobactams, are the most widely used antibiotics worldwide. In fact, approximately two-thirds of prescribed antibiotics are β-lactam antibiotics [3]. Similar to other antibiotics, the extensive use of β-lactam antibiotics has led to the development of resistance. In terms of β-lactam antibiotics, the main resistance arises from the degradative enzymes, β-lactamases [4]. The β-lactam ring of β-lactams resembles the _D-Ala _D-Ala moiety, and the direct binding to PBPs inhibits bacterial cell wall synthesis [5]. β-lactamases hydrolyze the β-lactam ring and nullify the antibiotic activity.

Two systems are traditionally used to classify β-lactamases: the Bush-Jacoby-Medeiros activity-based system [6], which is based on functional characteristics of β-lactamases; and the Ambler system [7], which is based on the protein sequence. According to specific amino acid motifs, the β-lactamases are classified into four classes: A, B, C, and D. Classes A, C, and D include serine-β-lactamases (SBLs) while class B includes metallo-β-lactamases (MBLs). OXA enzymes, classified as class D based on their amino acid identity, effectively hydrolyze oxacillin, as implied by their names [8]. OXA enzymes are predominantly detected in various pathogenic bacteria, particularly P. aeruginosa [9,10]. P. aeruginosa, a highly adaptive Gram-negative pathogen, is a major cause of nosocomial infections, presenting significant challenges in hospital environments [10,11].

OXA genes are located on chromosomes and plasmids with mobile elements, such as transposons for mobilization [12]. Through a plasmid-mediated transfer system, OXA enzymes can diffuse worldwide and acquire the ability to hydrolyze new antibiotics [13]. This phenomenon is particularly concerning for oxyiminocephalosporins and carbapenems, which are regarded as last-resort treatments for combating multidrug-resistant (MDR) infections [14,15].

OXA enzymes are primarily categorized into three classes based on their substrate specificities [16]. The first group comprises narrow-spectrum enzymes that hydrolyze penicillins (ampicillin and benzylpenicillin) and narrow-spectrum cephalosporins (cephalothin and cephaloridine), including OXA-1, OXA-2, and OXA-10 [16,17]. The second group consists of expanded-spectrum enzymes that can hydrolyze broad-spectrum cephalosporins (cefotaxime, ceftazidime, and cefepime), with OXA-14, OXA-32, and OXA-45 as representatives [18,19]. The final group contains the carbapenem-hydrolyzing enzymes, which confer resistance to carbapenems (imipenem, meropenem, and doripenem) and include OXA-23 and OXA-48 [20].

The second group of OXA enzymes is further subdivided into two main types: point mutation derivatives of narrow-spectrum enzymes and completely different enzymes with different amino acid sequences [16]. For the point-mutation derivatives, the substrate spectrum is expanded by several mutations in group 1, leading to an increased hydrolysis rate of broad-spectrum cephalosporins. Narrow-spectrum enzymes may thus have the evolutionary potential to develop into powerful and clinically important variants [21]. Among group 1 enzymes, OXA-10 possesses the most point mutation derivatives with enhanced hydrolytic activity against broad-spectrum cephalosporins [16].

OXA-10-dependent drug-resistant P. aeruginosa has been increasingly reported in clinical settings worldwide. Initially identified in Turkey, where the first strain of ceftazidime-resistant P. aeruginosa expressing OXA-10 variants was detected [8], the resistance mechanism has since spread to several regions in Europe, particularly France, as well as the Middle East and North Africa [22-24]. Studies from Iran, Saudi Arabia, and Iraq have demonstrated a high prevalence of OXA-10-producing P. aeruginosa in hospital environments, such as burn units and intensive care settings [24,25]. In addition, nosocomial isolates of P. aeruginosa expressing OXA-10 variants have been reported in tertiary care centres in Northeast India [9]. In Nigeria, the first detection of OXA-10 in Africa marked a significant expansion of this resistance gene beyond its original regions, such as Turkey and France [23]. The occurrence of OXA-10 variants, including OXA-145, has been highlighted in areas such as France (Indian Ocean), where they contribute to resistance against 3^rd cephalosporins [22]. Overall, the spread of OXA-10-related resistance has now been observed across multiple continents, including Asia and Africa, underscoring the global nature of this resistance mechanism [12].

In this study, we hypothesize that single-point mutations in the OXA-10 can significantly broaden its substrate spectrum, particularly enhancing its hydrolytic activity against cephalosporins. Through structural and biochemical analysis, we expect specific point mutations to increase hydrolytic efficiency against ceftazidime. This would provide evidence of the evolutionary potential of narrow-spectrum β-lactamases like OXA-10 to expand their substrate spectrum with minimal genetic alterations. The validation of this hypothesis will deepen our understanding of how structural changes affect substrate recognition in β-lactamases, providing critical insights into drug resistance mechanisms.

Materials and methods

Reagents

The expression vector, pET-29b, was purchased from Novagen (San Diego, CA, USA). The expression host, E. coli BL21(DE3), and all restriction enzymes were purchased from New England Biolabs (Hertfordshire, UK). Luria–Bertani (LB) medium was purchased from BD Biosciences (San Jose, CA, USA). Pre-stained protein markers for SDS-PAGE and the gel filtration calibration kit were purchased from MBI Fermentas (Hanover, MD, USA) and GE Healthcare (Piscataway, NJ, USA), respectively. The β-lactam antibiotics were purchased from different companies: ceftazidime from Tokyo Chemical Industry (Tokyo, Japan), and ampicillin from Duchefa Biochemie (Haarlem, Netherlands).

Plasmid construction and cloning

The DNA template encoding the OXA-10 gene from P. aeruginosa was amplified via PCR using a forward primer (5’-CCC CCC CAT ATG TCA ATT ACC GAA AAC ACG-3’) and a backward primer (5’-CCC CCC CTC GAG CTA TTA CCC ACC AAT GAT – 3’); the restriction sites (NdeI and XhoI, respectively) are highlighted in bold text. The amplified DNA fragments and the pET-29b vector were double-digested with NdeI and XhoI. The pET-29b vector was modified to contain additional residues of a 7×His-tag and a Tobacco etch virus (TEV) protease cleavage site (SSENLYFQGH) before the NdeI site.

Site-directed mutagenesis was performed using PCR to generate the OXA-10 variants, G157D, A124T, and N73S. The digested DNA fragments were ligated into the pET-29b vector, and the resulting plasmid was transformed into E. coli DH5α^TM cells. The cells were grown in LB medium containing 50 μg/mL kanamycin at 37 ℃.

Expression

After verifying the DNA sequences, plasmids were individually transformed into E. coli BL21(DE3) cells. As performed for E. coli DH5α^TM cells, E. coli BL21(DE3) cells were grown in 50 μg/mL of kanamycin-containing LB medium at 37 ℃. Grown cells were inoculated into 8 L of LB liquid medium (50 mg/mL of kanamycin) at 37 ℃. When an OD_{600 nm} of 0.6 was achieved, 0.5 mM isopropyl β-_D-1-thiogalactopyranoside (IPTG) was added to the culture to induce the expression of OXA-10 and its variants. After 24 h of culture at 15 ℃ for enzyme expression, the cells were harvested via centrifugation at 50,000×g for 30 min at 4 ℃ using a Vision VS24-SMTi V506A rotor.

Protein purification

The cells were resuspended in ice-cold lysis buffer (25 mM Tris-HCl pH 8.5, 300 mM NaCl, 20 mM imidazole, 3 mM β-mercaptoethanol, 10% glycerol), disrupted using a sonicator (Sonomasher) in an ice bath, and centrifuged at 72,400×g for 90 min at 4 ℃ using a Vision VS24-SMTi V508A rotor to remove the cell debris. The clarified supernatant was loaded onto a HisTrap Ni-NTA column (GE Healthcare) pre-equilibrated with lysis buffer. Thereafter, His-tagged OXA-10 and its variants were eluted with a linear gradient of imidazole (20-250 mM) in the elution buffer (25 mM Tris-HCl pH 8.5, 300 mM NaCl, 250 mM imidazole, 3 mM β-mercaptoethanol, 10% glycerol). The His-tag was removed from for further purification using TEV protease. The reaction was allowed to proceed for 16 h at 10 ℃, along with desalination. The reaction mixture was concentrated using a centrifugal filter (Amicon ® Ultra-15, MWCO 10 kDa). Thereafter, the sample was loaded onto a HiTrap Q anion-exchange column (GE Healthcare) pre-equilibrated with buffer A (25 mM Tris-HCl pH 8.5, 3 mM β-mercaptoethanol, 10% glycerol). The enzyme, which binds to cation resins, was eluted with a linear gradient of NaCl (0-1 M) in buffer B (25 mM Tris-HCl pH 8.5, 3 mM β-mercaptoethanol, 1 M NaCl, 10% glycerol). All purification steps were analysed using SDS-PAGE. The samples were concentrated to 10 mg/mL using a centrifugal filter (Amicon ® Ultra-15; MWCO, 10 kDa). The protein concentration was measured using the Bradford protein assay.

Enzyme assay

A discontinuous enzyme kinetics study of purified OXA-10 and its variants was performed using a modified spectrophotometric method to estimate their enzymatic activities. The absorbance of ampicillin and ceftazidime was measured using an Infinite M200 Pro (TECAN, Männedorf, Switzerland) at room temperature (∼25 ℃) in a UV-transparent 96-well plate (Corning ®, Corning, NY, USA).

The characteristic maximum absorption of each antibiotic was used to determine the concentration; a wavelength of 235 nm was used for ampicillin while 260 nm was used for ceftazidime. All substrate stock solutions were diluted to working concentrations ranging from 0 to 1 mM for ampicillin and 0–0.4 mM for ceftazidime. Thereafter, 190 μL of the assay buffer (25 mM Tris-HCl pH 8.5) was mixed with 10 μL of each substrate solution, and standard curves were generated. The linearity between the absorbance (absorption maximum of each antibiotic) and the antibiotic concentration was used as a standard to measure the antibiotic concentration. Reliable correlations (ampicillin: R² = 0.9996; ceftazidime: R² = 0.9989) were obtained by curve fitting using linear regression analysis (Figure S1). OXA-10 and its variants were diluted with assay buffer, and their concentrations were measured using the Bradford method, with bovine serum albumin (BSA) as the standard (from 0 to 1 mg/mL). Subsequently, 180 μL of assay buffer (25 mM Tris-HCl pH 8.5), 10 μL of each concentration of substrate solutions, and 10 μL of enzymes were mixed. The absorbance was recorded every 30 s at room temperature, and the change in time-dependent absorption (Figures S2 and S3) was used to calculate the hydrolysis rate of the antibiotics at a specific antibiotic concentration. The kinetic parameters were determined by fitting the data to the Michaelis–Menten equation.

Crystallization

Purified wild-type OXA-10 and its variants were crystallized at 15 ℃ using the Hydra II e-drop automated pipetting system (Matrix) on a 96-Well Intelliplate (Art Robbins) and Hampton research buffer sets. Drops consisted of 0.5 μL protein solution and 0.5 μL reservoir solution, and were equilibrated against 50 μL of reservoir solution at 15 ℃. After obtaining the initial conditions for crystal formation, optimization was performed using the hanging-drop vapour diffusion method in a 24-well plate (SPL Life Sciences, Pocheon, Republic of Korea). The crystallization reservoir solution contained 0.1 M Tris-Bicine pH 7-9, 16-22% (w/v) PEG 3350, and 0.2 M Sodium formate. Each of the drops contained 1 μL of protein solution and 1 μL of reservoir solution. The crystals were initially observed within 3 days and were fully grown for 2 weeks. The fully grown single crystals were transferred to a mixture of reservoir solution and cryoprotectant solutions (30% glycerol), with or without ceftazidime. The crystals were flash-cooled at – 173 ℃ in liquid nitrogen before data collection.

Data collection, structure determination, and refinement

X-ray diffraction data were collected on the beamline 11C at the Pohang Light Source (PLS) in South Korea. Diffraction data were integrated and scaled using the DENZO and SCALEPACK algorithms, respectively. The initial stage of the structure was determined by molecular replacement (MR) using the Phaser program of the CCP4 8.0.008 software package, and OXA-10 (PDB ID: 1FOF) was used as a search model for phaser MR. OXA-10 residues and their variants were manually built using the COOT program with multiple cycles of Refmac5 of the CCP4 software package for automated refinement. Data collection and structural refinement statistics are presented in Table S1. Multiple alignments of protein sequences were performed using Clustal Omega and presented using ESPript 3. Graphical presentations were created using the PyMOL 4.6 software.

CD spectroscopy

The CD spectra were recorded using a Jasco J-1700 instrument. Enzymes were prepared at a concentration of 0.1 mg/mL in 10 mM sodium phosphate buffer (pH 7.0) and measured in a cuvette with a 1 mm pathlength. Spectra were collected over a wavelength range of 185–260 nm with 0.5-nm increments, and each data point was averaged for 1 s.

Results

Substrate-binding pocket and β-lactam recognition of OXA-10

The substrate-binding pocket of OXA-10 was divided into four major loops, loop 1 to loop 4 (L1 to L4), in a counterclockwise direction from the right to clarify its structural environment (Figure 1A). L1 was found between α3 and α5; L2, usually known as the omega loop [8,22], was between α5 and α6; L3, also known as the β5-β6 loop in class D carbapenemases [8,26], was between β7 and β8; and L4 was between β9 and α8 (Figure S4). The catalytic serine residue was located at the centre of these four loops. When OXA-10 was superimposed with β-lactam antibiotics, the side chain in the β-lactam ring (R_β), which is shaded red, was identified to bind to the upper side of the substrate-binding pocket between L2 and L3, while that in the fused additional ring (R_a), which is shaded blue, bound to the lower side of the substrate-binding pocket between L1 and L4 (Figure 1B).

Figure 1.

(A) Superimposed structure of OXA-10 (PDB ID: 9IXN) in complex with ceftazidime (PDB ID: 4X55). eL1, eL2, eL3, and eL4 are marked in cyan, magenta, orange, and yellow, respectively. The side chain in the β-lactam ring (R_β) is shaded red while that in the fused additional ring (R_a) is shaded blue. (B) Schematic of OXA-10 bound by the β-lactam antibiotic. Structurally corresponding parts in the OXA-10 and β-lactams are indicated in the same colours used in (A). (C) Chemical structures of penicillin and narrow – and broad-spectrum cephalosporins. The side chains in R_β and R_a are shaded using the same colours in (A).

Structural compatibility between substrate-binding pocket and β-lactams

OXA-10, known as a narrow-spectrum β-lactamase, efficiently hydrolyzes penicillins and 1^st and 2^nd generation cephalosporins, so-called narrow-spectrum cephalosporins [16]; however, this enzyme exhibits limited hydrolytic activity for 3^rd and 4^th generation cephalosporins. The broad-spectrum cephalosporins were distinguished by their large side chain in R_β compared to penicillins and narrow-spectrum cephalosporins (Figure 1C).

To determine the impact of the size difference in the side chain in R_β between penicillins and broad-spectrum cephalosporins on OXA-10, ampicillin with a smaller side chain and ceftazidime with a large side chain were selected as representative β-lactams of penicillins and broad-spectrum cephalosporins, respectively. The ampicillin and ceftazidime-bound β-lactamase structures were superimposed onto the OXA-10 to understand the structural compatibility between the substrates and substrate-binding pocket of OXA-10 (Figure 2A). The structures revealed a significant difference in the distance between L2 and the side chain in R_β. Ceftazidime, characterized by its large side chain in R_β, had a closer proximity to L2 and exhibited steric clashes compared to ampicillin.

Figure 2.

(A) Structure-based sequence alignment of OXA-2 and OXA-10. The figure was produced using the ESPript 3.0 program. Identical residues among all amino acid sequences are indicated in black. The cyan, magenta, orange, and yellow lines indicate eL1, eL2, eL3, and eL4, respectively. The mutation residues within L2 are shown in a circle filled with magenta and those surrounding L2 are shown in a circle filled with black.

The enzyme kinetics of OXA-10 was studied using ampicillin and ceftazidime (Figure 2B). OXA-10 exhibited higher hydrolytic activity against ampicillin than ceftazidime. The enzyme kinetic results were consistent with the structural compatibility between the substrates and substrate-binding pocket, as shown in Figure 2A.

Key point mutations in ES-OXA variants

OXA-10 exhibited weak hydrolytic activity against ceftazidime, a broad-spectrum cephalosporin. Several OXA variants gained the ability to hydrolyze the broad-spectrum ceftazidime through point mutations and become ES-OXAs (expanded-spectrum class D β-lactamases). The OXA-2 and OXA-10 groups of variants induced increased resistance to broad-spectrum cephalosporins with single-point mutations, almost all of which existed in L2 (Table 1). Variants with multiple point mutations commonly had mutations in L2. In the OXA-10 group, ES-OXAs frequently had the G157D substitution. Among ES-OXAs, only OXA-17 and OXA-35 lacked mutations in L2. Although OXA-35 did not show increased activity against broad-spectrum cephalosporins, OXA-17, which has a single-point mutation in the N73S mutation, showed broad-spectrum activity, unlike OXA-35.

Table 1.

Mutation residues and substrate spectrum of ES-OXAs.

Narrow-spectrum	Expanded-spectrum	Mutation residuesa	Mutations in L2b	Broad-spectrum cephalosporinsc
OXA-2	OXA-15	D150G	O	↑d
	OXA-32	L164I	O	↑e
	OXA-53	R4Q, I12T, S14 V, E23D, R32G, E37D, R54H, M56I, P61Q, V62A, K65M, N101K, G103S, Q109K, D135G, K143Q, D145G, N155H, R180Q, E182D, R216S, S230P, V253A, R258L, N270H	O	↑f
	OXA-141	G162S	O	↑g
	OXA-161	N148D	O	↑h
OXA-10	OXA-11	N143S, G157D	O	↑i
	OXA-14	G157D	O	↑j
	OXA-16	A124T, G157D	O	↑k
	OXA-17	N73S	X	↑l
	OXA-19	I10T, G20S, D55N, T107S, G157D, Y174F, E229G, S245N, E259A	O	↑m
	OXA-28	I10T, G20S, D55N, T107S, W154G, Y174F, E229G, S245N, E259A	O	↑n
	OXA-35	I10T, G20S, D55N, T107S, Y174F, E229G, S245N, E259A	X	─o
	OXA-142	N73S, G157D	O	↑p
	OXA-145	I10T, G20S, D55N, T107S, L155, Y174F, E229G, S245N, E259A	O	↑q
	OXA-147	I10T, G20S, D55N, T107S, W154L, Y174F, E229G, S245N, E259A	O	↑r
	OXA-935	F153S, G157D	O	↑s

^aMutation residues located in L2 are highlighted in bold and magenta.

^bES-OXAs with point mutations in L2 are indicated by “O,” while those without such mutations are indicated by “X”.

^cEnzymes exhibiting increased resistance to broad-spectrum cephalosporins compared to narrow-spectrum enzymes are indicated by “↑”, whereas those with similar resistance to narrow-spectrum enzymes are denoted by “─”.

^dRef. [32].

^eRef. [19].

^fRefs. [16,33].

^gRef. [34].

^hRef. [35].

ⁱRef. [36].

^jRef. [18].

^kRef. [27].

^lRef. [28].

^mRef. [37].

ⁿRef. [38].

^oRef. [39].

^pRef. [40].

^qRef. [22].

^rRef. [41].

^sRef. [29].

As numerous point mutations complicate the identification of specific residues that affect substrate spectrum changes, variants with one or two point mutations that increase the ability to hydrolyze broad-spectrum cephalosporins were studied (Figure 3). In the OXA-2 group, all variants except OXA-53, and in the OXA-10 group, all variants except OXA-19, OXA-28, OXA-145, and OXA-147, were compared to identify key point mutations.

Figure 3.

(A) Structure-based sequence alignment of OXA-2 and OXA-10. The figure was produced using the ESPript 3.0 program. Identical residues among all amino acid sequences are indicated in black. The cyan, magenta, orange, and yellow lines indicate eL1, eL2, eL3, and eL4, respectively. The mutation residues within L2 are shown in a circle filled with magenta and those surrounding L2 are shown in an empty magenta circle. Structurally corresponding parts in OXA-2 (PDB ID: 1K38) (B) and OXA-10 (C) are indicated in the same colours used in (A). The mutation residues are yellow-orange in structures.

Most point mutations were concentrated in the L2 region (Figure 3A). Notably, the N148D and G162S substitutions in the OXA-2 group occupied structural positions similar to the N143S and G157D substitutions in the OXA-10 group, respectively. Although most mutations occurred in L2, some occurred outside L2. The N73S mutation in OXA-17, which alters substrate specificity, resided outside L2 but was identified to be structurally proximal to L2 (Figure 3C). Similarly, the A124T mutation coexisting with the G157D mutation in OXA-16 also resided outside L2 but was next to L2. Based on these observations, three key residues were identified for the transition from narrow-spectrum OXA to ES-OXA: the G157D mutation, which was frequently observed in the OXA-10 group and occupied a structurally analogous position to the G162S mutation in the OXA-2 group; the N73S mutation, which conferred resistance to broad-spectrum cephalosporins despite not being in L2; and the A124T mutation, which was near L2, similar to N73S. We proceeded to assess the three variants with the G157D, N73S, and A124T mutations.

Structural verification of key point mutations for transition to ES-OXAs

Although the G157D, A124T, and N73S substitutions were distributed across the entire protein sequence (Figure S4), they were structurally concentrated around L2 (Figure 4). The structures of the wild-type enzyme and three variants were compared to determine the structural changes induced by point mutations. The G157D substitution, located within L2, induced widening of L2 compared to the size of the wild-type structure. The A124T substitution, located adjacent to L2 and possessed a bulkier side chain, resulted in an upward shift of I150 in L2. The N73S substitution, which was below L2 and had a reduced side chain, increased the distance from W154 in L2.

Figure 4.

Structural details of the mutation sites in OXA-10. The eL1, eL2, eL3, and eL4 are marked in cyan, magenta, orange, and yellow, respectively. In enlarged figures, wild-type OXA-10 is indicated in white while the variants are indicated in magenta. The mutation residues are coloured yellow-orange in variants. The side chains of the 3 mutations are depicted using sticks and marked as red circles. The dotted red circle indicates the disappeared side chain of residue S73. I150 and W154 are depicted by sticks. The impact of point mutations is indicated using red arrows. PDB IDs are as follows: G157D variant (9IXO), A124T variant (9IXP), and N73S variant (9IXQ).

The OXA-10 variants exhibited hydrolytic activity similar to or lower than that of the wild-type OXA-10 for ampicillin; however, all variants exhibited increased hydrolytic activity for ceftazidime (Figure 5B).

Figure 5.

(A) Overall mutation locations in the OXA-10 variants. eL1, eL2, eL3, and eL4 are marked in cyan, magenta, orange, and yellow, respectively. The sites of three-point mutations are coloured yellow-orange and marked as red circles. Two dotted red circles on the surface indicate that their location on the inside. (B) Hydrolysis activities of wild-type OXA-10, and the G157D, A124T, and N73S variants to ampicillin and ceftazidime according to their several concentrations. The chemical structure of each antibiotic is shown in the top left corner of the graph. Wild-type OXA-10 is indicated by an empty black circle, while the G157D, A124T, and N73S variants are depicted by a triangle, diamond, and circle filled with red, respectively. All tests were repeated three times, with error indicated by an error bar.

Conformational changes of L2 upon ceftazidime binding

By comparing the complex structures of the A124T variant bound to ceftazidime with its apo structure, L2 was recognized to undergo substantial conformational changes. However, the other three loops in the substrate-binding pocket remained unchanged (Figure 6A,B). Ceftazidime induced steric hindrance between the side chain in R_β and L2, causing a 1.5 Å outward shift of L2.

Figure 6.

(A) Superimposition of the apo structure of the A124T variant and the variant complex bound by ceftazidime (PDB ID: 9IXR), shown in white and magenta, respectively. The mutation residue is coloured yellow-orange in the variant. The movement of L2 is indicated by the red arrow. (B) Schematic of the A124T variant bound by ceftazidime. Structurally corresponding parts in the A124T variant and β-lactams are marked in the same colours as (A). The A124T mutation site is shown in a circle filled with yellow-orange. The red arrow represents the expected movement of L2. (C) The FoFc electron density maps contoured at 2.7 e/ų are shown green mesh for A124T variants. The side chains of the A124T mutations are represented as sticks marked in yellow-orange.

In the FoFc omit electron density maps contoured at 2.7 e/ų, the electron densities for the conserved central core α3, used as a reference, were similar between apo and complex structure. However, the variant showed a decrease in the electron density of L2 in the complex structure compared to that in the apo structure (Figure 6C). These results indicate that L2 is the most variable part among the four loops during binding of β-lactam antibiotics with large side chains in R_β.

Increased flexibility of L2 in the OXA-10 variants

We compared the crystal structure of wild-type OXA-10 with three variants using FoFc electron density maps contoured at 2.7 e/ų, where the difference between the wild-type and variants was clearly distinguishable (Figure 7A,B). The conserved central core α3 showed similar electron densities between wild-type and variants, while those for L2 showed significant differences. In the G157D variant, the electron density of L2 was markedly lower than that in the wild-type structure. In the A124T and N73S variants, the electron density of L2 decreased mainly around the mutated residue compared with that in the wild-type structure. These reductions in electron density in the variants indicate that L2 became more flexible after a single amino acid substitution (Figure 7C).

Figure 7.

(A) The overall structure of OXA-10. The eL1, eL2, eL3, and eL4 are marked in cyan, magenta, orange, and yellow, respectively. The mutation residues are coloured yellow-orange. The sites where the FoFc map is shown in (B) are marked as black boxes. (B) The FoFc electron density maps contoured at 2.7 e/ų are shown as green mesh for wild-type and variants. The side chains of the mutations are represented as sticks marked in yellow-orange. (C) The schematic drawing of OXA-10 with mutation sites. Structurally corresponding parts in the OXA-10 are indicated in the same colours used in (A). The dotted magenta line indicates the expected appearance of L2 in the three variants.

To investigate the effects of single amino acid substitutions on the overall secondary structure of OXA-10, CD spectroscopy was conducted (Figure S5). The CD spectra for all three variants showed noticeable differences compared to the wild-type, indicating conformational changes. Among the variants, the G157D mutation located in L2 exhibited the largest deviation from the wild-type spectrum. This suggests that point mutation in L2 induces more significant structural changes, particularly in the local flexibility of L2. Overall, these results indicate that the three-point mutations affect the conformation of OXA-10, primarily through localized changes in L2, while maintaining the overall secondary structure.

The increased flexibility of L2 was further supported by the analysis of B factors. Compared with the wild-type OXA-10, L2 of the OXA-10 variants generally had higher B factors (Table S2). The ratio of the B factors between L2 and the stable inner core scaffold provides insights into the flexibility of L2. The ratio of B factors between L2 and other regions was consistently higher in the OXA-10 variants than in the wild-type. Based on their B factor values, OXA-10 variants had thicker and redder L2 regions around the mutated residues than the wild-type (Figure S6A–D). This result suggests that L2 in OXA-10 variants is more flexible than that in wild-type OXA-10.

Discussion

In ES-OXAs, point mutations expand their substrate spectra. Most ES-OXAs with increased hydrolytic activity towards broad-spectrum cephalosporins have at least one point mutation at L2 (Table 1). However, variants lacking L2 mutations do not exhibit this resistance. In the OXA-10 group, OXA-35, which lacks any point mutations in L2, showed hydrolytic activity similar to that of OXA-10 for broad-spectrum cephalosporins. In contrast, the variants OXA-19, OXA-28, OXA-145, and OXA-147, each with a single additional point mutation in L2 compared with OXA-35, exhibited increased resistance to broad-spectrum cephalosporins. Point mutations in L2 confer resistance to broad-spectrum cephalosporins in ES-OXAs.

Among the various point mutations, we selected G157D, A124T, and N73S mutations. First, we focused on L2 where most of the point mutations are clustered (Table 1 and Figure 3A). The G157D mutation commonly occurs in OXA-10 variants, including OXA-11, OXA-14, OXA-16, OXA-19, OXA-142, and OXA-935. It is also structurally analogous to the G162S mutation found in OXA-161, a variant of OXA-2. Given its prevalence in the OXA-10 group and its location in L2, we chose the G157D mutation for further study. After selecting a mutation within L2, rather than opting for another mutation in the same region, such as F153S or W154G, we turned our attention to mutations outside of L2. The N73S mutation in OXA-17, although located adjacent to L2, enhances the hydrolytic activity against broad-spectrum cephalosporins. To better understand how this mutation affects substrate specificity at a structural level, we selected the N73S mutation. Similarly, we chose the A124T mutation, another mutation outside of L2. The A124T mutation coexists with the G157D mutation in OXA-16 and near L2, and we aimed to determine whether the A124T mutation alone can alter substrate specificity and how it influences the enzyme’s function.

These point mutations in L2 increase the flexibility of L2, enabling the hydrolysis of broad-spectrum cephalosporins with a large side chain in R_β. The G157D variant, which is located within L2, exhibited hydrolytic activity similar to that of the wild-type for ampicillin, but had a higher activity for ceftazidime, with a significant reduction in L2 electron density in the FoFc map. Mutations in the regions surrounding L2 also expanded the substrate spectrum. The A124T and N73S variants had higher hydrolytic activity towards ceftazidime, with decreased electron density in L2. All three substitutions enhanced the flexibility of L2, facilitating the binding of ceftazidime, which has a large side chain in R_β.

CD spectroscopy analysis highlighted notable structural differences between the wild-type and its variants, which aligned with data from the B factor ratio (Table S2) and the FoFc electron density maps (Figure 7B). The G157D variant, which exhibited the highest B factor ratio as well as the largest reduction in electron density, also showed the greatest difference from the wild-type in CD spectroscopy. The A124T and N73S mutations, both located near L2, displayed similar B factor ratios, with the N73S variant showing a slightly greater reduction in electron density compared to the A124T variant. Correspondingly, CD spectra revealed that the N73S variant exhibited a more pronounced deviation from the wild-type than the A124T variant. These results suggest that while all three mutations increase the flexibility of L2, point mutations in L2, such as the G157D, induce the most significant structural changes, followed by those near L2, such as N73S and A124T.

These results highlight that L2 and its adjacent regions may serve as critical hotspots conferring the ability to hydrolyze broad-spectrum cephalosporins by affecting the flexibility of L2. In particular, the region between α5 and α6 in OXA-10, corresponding to L2; the α5 region, representing the right side of L2; and the α3 region, representing the bottom part of L2, could be identified as such hotspots (Figure 3A,C). Although not annotated with mutations in Figure 3A, the region between α2 and α3, corresponding to the left side of L2, could also serve as a hotspot.

Point mutations in L2 and its surrounding regions play a critical role in enhancing resistance to broad-spectrum cephalosporins in clinically relevant OXA-10 variants. The G157D mutation, present in 6 of the 11 OXA-10 variants in Table 1 (approximately 54%), is critical for conferring resistance to ceftazidime. Specifically, OXA-14, which carries the G157D mutation and was isolated from patients at Hacettepe University Hospital in Ankara, Turkey, exhibited a 128-fold increase in the MIC for ceftazidime relative to OXA-10 [18]. Similarly, OXA-16, which has both the G157D and A124T mutations in OXA-10, also isolated from the same hospital, exhibited a 128-fold increase in the MIC for ceftazidime compared to OXA-10 [27]. OXA-17, carrying the N73S mutation, was likewise isolated from the same hospital and exhibited a 4-fold increase in the MIC for ceftazidime compared to OXA-10 [28]. Despite these differences, all three variants indicated that the MICs for ampicillin were either similar to or up to 2-fold lower than that of the wild-type enzyme but retained resistance to penicillins. These results confirm that such point mutations confer resistance to broad-spectrum cephalosporins while maintaining the hydrolytic activity of penicillins.

In our study, we observed that mutations in L2 and its adjacent areas increased the flexibility of L2, facilitating the binding of substrates with a bulky side chain in R_β such as broad-spectrum cephalosporins. This was consistent with previous findings, where the F153S and G157D substitutions in OXA-935 enhanced the flexibility of L2, allowing the enzyme to accommodate substrates with large side chains like ceftazidime [29]. In OXA-935, the F153S and G157D mutations induce a more flexible L2, with K70 in a decarbamylation state, preventing the formation of a hydrogen bond with W154 (16.4 Å). This structural change enlarges the active site, improving substrate accessibility. However, in contrast to this previous study, our crystal structures of both wild-type OXA-10 and its three variants revealed that K70 remained carbamylated in all cases, maintaining a stable hydrogen bond with W154 (3 Å). This difference suggests that while a single-point mutation in L2 is sufficient to increase its flexibility, the presence of two or more point mutations in L2, as observed in OXA-935, may result in a more open active site due to K70 decarbamylation. Despite these structural differences, the increased flexibility in L2 plays a critical role in expanding the substrate spectrum, particularly towards bulky cephalosporins.

In the structures coloured according to the B factor values (Figure S6A–D), L3 appeared red and thicker in both wild-type and variants, indicating its inherent flexibility regardless of the enzyme variant. This region is located on the outer periphery of the enzyme, away from the substrate binding site. This finding was supported by the ceftazidime-bound complex structure in which minimal movement of L3 was observed before and after substrate binding (Figure 6A). Long L3 is a characteristic feature of non-carbapenemases, such as OXA-10. In contrast, most class D carbapenemases, such as OXA-23 and OXA-48, have a short L3 positioned inwards within the substrate-binding pocket, forming a narrow active site cleft [30]. Inserting a single alanine into L3 of OXA-23 resulted in the ability to hydrolyze broad-spectrum cephalosporins [20]. The extended length of L3, which protrudes outward and enlarges the substrate-binding pocket, facilitates the binding of broad-spectrum cephalosporins with a large side chain in R_β. Therefore, the extended length and increased flexibility of L3 could play a crucial role in hydrolyzing broad-spectrum cephalosporins, such as L2.

Variants with point mutations that increase the flexibility of L2 facilitate the easier movement of L2, thereby enhancing the binding of broad-spectrum cephalosporins. In contrast to broad-spectrum cephalosporins, carbapenems have a larger side chain for R_a than penicillins, which bind to the lower side of the substrate-binding pocket between L1 and L4. Both narrow-spectrum β-lactamases and ES-OXAs are generally ineffective at hydrolyzing carbapenems. Thus, the side chain size, in terms of R_a, may be minimally affected by L2 flexibility. Additional mutations in L1 and L4 may change the bottom of the substrate-binding pocket to accommodate large side chains in R_a.

Broad-spectrum cephalosporins with a bulky side chain in R_β require a large upper space in the substrate-binding pocket for effective binding. Evolutionarily, a method was developed to accommodate this requirement by increasing the flexibility of L2. Point mutations in L2 and its surrounding regions can induce increased flexibility in L2, thereby conferring resistance to broad-spectrum cephalosporins. This enhanced activity provides a survival advantage to these variants and poses a greater threat to human health. Although existing β-lactamase inhibitors operate by either binding to the enzyme with high affinity or functioning as suicide inhibitors that permanently inactive the enzyme, there are no clinically approved inhibitors specifically targeting these mutations [31]. The substrate-binding pockets of β-lactamases having a flexible L2 are easily adjustable to bind various inhibitors. However, it may be possible to take advantage of the structural limits of L2’s flexibility. Inhibitors with a bulkier side chain, which may not fit into the open conformation of the substrate-binding pocket, could prevent the enzyme from adjusting and binding properly, thereby blocking its activity. Further studies are needed to verify the efficacy of such potential inhibitors.

Appendix.

Appendix A1.

Genbank accession numbers.

OXA enzyme	Accession number
OXA-2	AEZ05110
OXA-15	AAB05874
OXA-32	AAK58418
OXA-53	WP_063864223
OXA-141	ABQ15112
OXA-161	ACT09125
OXA-10	ACI28891
OXA-11	WP_063860939
OXA-14	WP_064056056
OXA-16	AAB97924
OXA-17	ABD58911
OXA-19	ACQ77169
OXA-28	AAF72942
OXA-35	ACR33071
OXA-142	AGX26771
OXA-145	ACN85419
OXA-147	WP_063861045
OXA-935	WP_141989064

Funding Statement

This work was supported by the Bio & Medical Technology Development Program of the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (NRF-2021R1A2C3004826 and NRF-2017M3A9E4078017) and by the Science Research Center (SRC) of the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2024-00407469).

Disclosure statement

No potential conflict of interest was reported by the author(s).