A Two Amino Acid Duplication, L167E168, in the Ω-Loop Drastically Decreases Carbapenemase Activity of KPC-53, a Natural Class A β-Lactamase

ABSTRACT

KPC-53 enzyme is a natural KPC variant which showed a duplication of L167E168 residues in the Ω-loop structure. The bla_KPC-53 gene was cloned both into pBC-SK and pET-24a vectors, and the recombinant plasmids were transferred by transformation in Escherichia coli competent cells to evaluate the antimicrobial susceptibility and to produce the enzyme. Compared to KPC-3, the KPC-53 was less stable and showed a dramatic reduction of k_cat and k_cat/K_m versus several β-lactams, in particular carbapenems. Indeed, a 2,000-fold reduction was observed in the k_cat values of KPC-53 for imipenem and meropenem. Concerning inhibitors, KPC-53 was susceptible to tazobactam and clavulanic acid but maintained resistance to avibactam. The molecular modeling indicates that the L167E168 duplication in KPC-53 modifies the interactions between residues involved in the catalytic pocket, changing the flexibility of the Ω-loop, which is directly coupled with the catalytic properties of the KPC enzymes.

INTRODUCTION

KPC-type carbapenemases are molecular class A serine-β-lactamases emerged in the mid-1990s in the United States (1), which exhibit a very broad substrate specificity, including most β-lactams. These enzymes, usually encoded by transferable plasmids, have experienced a global dissemination in Klebsiella pneumoniae and other Enterobacterales, providing a relevant contribution to the increasing prevalence of carbapenemase-producing Enterobacterales observed in several epidemiological settings (2–5). Due to the very broad substrate specificity of these enzymes, KPC-producing strains usually show resistance to older β-lactams, including carbapenems, and older β-lactamase inhibitor combinations (BLICs), while they are susceptible to novel BLICs based on inhibitors that are active against KPC (e.g., ceftazidime-avibactam [CZA], imipenem-relebactam, and meropenem-vaborbactam) (6).

To date, 108 KPC variants which differ from each other by one to five amino acid substitutions (http://www.ncbi.nlm.nih.gov/pathogens/isolates#/refgene/KPC) have been reported. Among them, KPC-2 and KPC-3 are the most prevalent (7).

Structural data have shown that, similar to other serine-β-lactamases, KPC enzymes contain two domains: one of the subdomains has an α-helical structure, whereas the other subdomain is represented by five β-sheets flanked by α-helical structures (8). The active site of KPC-2 includes several conserved residues which appear to be directly or indirectly involved in the substrate recognition and catalytic process. The active site contains the catalytic residue S70, typical of all class A β-lactamases, and some conserved residues such as K73, W105, N132, E166, R220, and K234. Residue E166 is involved in the deacylation step of catalysis, while residues W105 and R220, located on opposite sides of the active site, are involved with the recognition of substrates and inhibitors (9). Previous studies indicated that some amino acid substitutions associated with KPC variants may affect substrate specificity and susceptibility to inhibitors (10–12). In particular, some KPC variants (e.g., those with the D179Y substitution) are associated with resistance to CZA, which is currently among the front-line antimicrobial agents used to treat infections caused by KPC-producing K. pneumoniae (13–15).

KPC-53 is a recently described natural KPC variant, detected in a K. pneumoniae strain resistant to CZA, which exhibits a L167E168 duplication in the Ω-loop of the enzyme (16). The aim of this study was to characterize the kinetic profile of KPC-53 and to investigate by molecular modeling the structure-function relationships of this KPC variant.

RESULTS

The bla_KPC-53 gene, identified in K. pneumoniae LC-1825/18 (16), was cloned both in pBC-SK and pET-24a(+) vectors. The recombinant plasmid (pET24-KPC-53) was introduced into Escherichia coli BL21(DE3) and used to overexpress the KPC-53 enzyme, whereas pBC/KPC-53 was inserted by transformation in E. coli XL-1 to evaluate the impact of KPC-53 on antimicrobial susceptibility.

Both E. coli XL-1/pBC/KPC-53 and E. coli XL-1/pBC/KPC-3 showed low MIC values for carbapenems (0.25 and 0.125 mg/L for imipenem and meropenem, respectively), but these strains were resistant to ceftazidime (Table 1). Avibactam reduced the MIC of ceftazidime from 128 to 1 mg/L and from 16 to 4 mg/L in E. coli XL-1/pBC/KPC-3 and E. coli XL-1/pBC/KPC-53, respectively. Tazobactam was unable to restore the susceptibility of piperacillin in E. coli XL-1/pBC/KPC-3. The E. coli XL-1/pBC/KPC-53 showed MICs for piperacillin and piperacillin-tazobactam of 8 and 2 mg/L, respectively.

TABLE 1.

Antibiotic MICs against E. coli XL-1/pBC/KPC-53 compared to that of E. coli XL-1/pBC/KPC-3

β-Lactama	MIC (mg/L) against E. coli XL-1 (5 × 105 CFU/mL)
	XL-1/pBC/KPC-53	XL-1/pBC/KPC-3	XL-1/pBC-SK
PIP	8	256	0.25
TZP	2	64	0.25
AMX	8	64	0.25
AMC	4	16	0.25
CAZ	16	128	<0.06
CZA	4	1	<0.06
IPM	0.25	0.25	<0.06
MEM	0.125	0.125	<0.06

^aPIP, piperacillin; TZP, piperacillin-tazobactam; AMX, amoxicillin; AMC, amoxicillin-clavulanic acid; CAZ, ceftazidime; CZA, ceftazidime-avibactam; IPM, imipenem; MEM, meropenem.

KPC-53 was purified from a cell extract by two chromatographic steps, and the purity grade of the enzyme was estimated to be >95% by SDS-PAGE. The isoelectric points (pIs) of purified KPC-53 and KPC-3 were 6.3 and 6.7, respectively. To analyze structural changes among the enzymes, fluorescence spectroscopy was used. Upon excitation at 280 nm, both KPC-3 and KPC-53 exhibited a fluorescence emission maximum of ~345 nm. KPC-53 spectra showed a 50% decrease in fluorescence intensity with respect to KPC-3 (see Fig. S1 in the supplemental material). A fluorescence-based thermal stability assay, performed using 3 μM concentrations of each pure enzyme, showed that the Boltzmann T_m values for KPC-53 and KPC-3 were 52.8 and 64°C, respectively.

The kinetic constants of KPC-53 with several substrates were determined and compared to those previously reported for KPC-3 (17). A reduction in k_cat was observed with all substrates, while K_m was variably affected and k_cat/K_m values were generally decreased compared to KPC-3, except for ceftazidime (Table 2). Imipenem and meropenem were poor substrates of KPC-53, and to determine their K_m values the reporter substrate method was used (18). KPC-53 and KPC-3 were also tested against some inhibitors including clavulanic acid, tazobactam, and avibactam (Table 3). Clavulanic acid behaved as a competitive inhibitor for KPC-3, with a K_i value of 41 μM, whereas it behaved as a transient inactivator for KPC-53. In this case, the plot of kiAQ versus clavulanic acid concentration was linear, and the second-order acylation rate constants were k₊₂/K = 3 × 10² M⁻¹ s⁻¹ and k₊₃ = 0.0016 s⁻¹. Tazobactam was hydrolyzed by KPC-3 with a k_cat of 6 s⁻¹ and a k_cat/K_m value of 0.022 μM⁻¹ s⁻¹ (Table 2). Instead, tazobactam acted as a transient inactivator for KPC-53 with a k₊₂/K of 2 × 10² M⁻¹ s⁻¹ and a k₊₃ of 0.0004 s⁻¹. Avibactam behaved as a transient inactivator of KPC-53, with a k₊₂/K of 6.3 M⁻¹ s⁻¹ and a k₊₃ of 0.0008 s⁻¹. In contrast, the KPC-3 plot of ki versus the avibactam concentration was not linear, and the inhibition constants were k₊₃ = 0, k₊₂ = 74 s⁻¹, and K = 1.1 × 10⁻² M.

TABLE 2.

Kinetic constants of KPC-53 and KPC-3 toward some β-lactams

Substrate	Parameter	Mean ± SD		KPC-3/KPC-53 ratio
		KPC-3a	KPC-53
Benzylpenicillin	Km (μM)	144 ± 22	23 ± 2	6.3
	kcat (s−1)	708 ± 15	1 ± 0.1	708
	kcat/Km (μM−1 s−1)	4.92	0.04	123
Carbenicillin	Km (μM)	108 ± 10	3 ± 0.2	36
	kcat (s−1)	18 ± 1	0.18 ± 0.014	100
	kcat/Km (μM−1 s−1)	0.17	0.06	2.8
Cefazolin	Km (μM)	189 ± 25	383 ± 34	0.49
	kcat (s−1)	351 ± 9	21 ± 2	16.7
	kcat/Km (μM−1 s−1)	1.86	0.05	37.2
Ceftazidime	Km (μM)	100 ± 15	31 ± 2	3.2
	kcat (s−1)	1.4 ± 0.1	0.35 ± 0.02	4
	kcat/Km (μM−1 s−1)	0.01	0.01	1
Imipenem	Km (μM)	88 ± 6	0.3 ± 0.02	293
	kcat (s−1)	41 ± 2	0.015 ± 0.001	2733
	kcat/Km (μM−1 s−1)	0.46	0.05	9.2
Meropenem	Km (μM)	68 ± 3	0.76 ± 0.06	89.5
	kcat (s−1)	51 ± 2	0.024 ± 0.001	2125
	kcat/Km (μM−1 s−1)	0.75	0.03	25
Tazobactamb	Km (μM)	277 ± 12	PSc	NDd
	kcat (s−1)	6 ± 1	PS	ND
	kcat/Km (μM−1 s−1)	0.022	PS	ND
Nitrocefin	Km (μM)	138 ± 10	106 ± 10	1.3
	kcat (s−1)	72 ± 5	4 ± 0.4	18
	kcat/Km (μM−1 s−1)	0.52	0.04	13

^aValues are expressed as means ± the standard deviations where applicable.

^bKPC-3 kinetic data were obtained from reference 17. The kinetic constants of KPC-3 against tazobactam were determined in this study.

^cPS, poor substrate.

^dND, not determined.

TABLE 3.

Inactivation of KPC-53 and KPC-3 by avibactam, tazobactam, and clavulanic acid

Inhibitor	KPC-53		KPC-3a
	k+3 (s−1)	k+2/K (M−1 s−1)	k+2 (s−1)	k+3 (s−1)	k+2/K (M−1 s−1)	K (M)	Ki (μM)
Avibactam	0.0008	6.3	74	0	6.8 × 103	1.1 × 10−2	ND
Tazobactam	0.0004	2 × 102	–	–	–	–	–
Clavulanic acid	0.0016	3 × 102	ND	ND	ND	ND	41

^a–, acted as a substrate; ND, not determined.

Molecular modeling.

Comparative protein structure molecular modeling was performed to explain the dramatic changes in the catalytic parameters observed in KPC-53 versus KPC-3. The three-dimensional (3D) structure of KPC-53 was obtained by comparing its aminoacidic sequence with the known 3D structures KPC-2 (PDB 5MGI) and KPC-3 (PDB 6QWD). The preliminary 3D structure of KPC-53 obtained by the satisfaction of spatial restraints, including the catalytic water molecule, was subsequently optimized for the loop regions, generating five plausible structures. The calculated scores for all generated models of KPC-53 indicated the reliability of the optimized structures. For example, the model score GA341, which assesses the quality of the model based on the percentage of sequence identity between the known 3D structure and the target, was 1 for all generated structures (a value higher than 0.7 is suggestive of a good prediction). The statistical potential z-DOPE (expected value lower than 0) for all structures were about −1.5 (ranging from −1.47 to −1.69). The last score is represented by the predicted root-mean-squared deviation (RMSD) between the spatial coordinates of the backbone carbon atoms in the model and the native structure. For all generated structures, the RMSD values were below 1 (ranging from 0.95 to 0.96) (see Table S1 in the supplemental material). All simulated KPC-53 structures are characterized by an α-helix, composed of five amino acids extending from E168 to N172 (N170 in KPC-2 and KPC-3), which partially overlays the short α-helix (3 amino acids from E168 to N170) commonly found in KPC-2 and KPC-3. The distance between the Oδ of N170 (N172 in KPC-53) for all simulated structures and the catalytic water ranges between 2.63 and 2.99 Å, allowing the formation of a hydrogen bond, as observed in all class A serine-β-lactamases. The L167E168 insertion distorts the carbon backbone between the W165 and the E166 (E168 in KPC-53), located in KPC-53, just before the ordered α-helix (L167-S171). The Cα of this glutamic residue lays from 0.463 to 0.831 Å apart and in any direction if compared to the Cα of E166 in KPC-2 crystallographic structure. The distance between the same carbon atom in KPC-2 and KPC-3 structures is only 0.06 Å. The distance between the W165 Cα is 0.05 Å between KPC-2 and KPC-3 but ranges from 0.30 to 0.90 Å in KPC-53 compared to KPC-2 (see Table S2). In all simulated models, E166 Oε is unable to interact via hydrogen-bond with the Nδ of N170 (N172 in KPC-53) except for one model where the distance is 2.79 Å, conceivable with a hydrogen-bond (see Table S3). As depicted in Fig. 1, the spatial coordinates of the backbone positions of all the residues involved in catalysis—S70, K73, S130, and K234 (K236 in KPC-53)—were found to be invariant.

FIG 1.

Superimposition of the five modeling-generated models of KPC-53 (orange) with the known crystallographic structure of KPC-2 (green) (PDB 5MGI). (A) Detail of the interactions between E166 of KPC-2 and the five models generated for KPC-53 in relation to the catalytic water molecule. (B) Detail of the deformation of the region between residues R164 and N170 of KPC-2 (green) and R164 and N172 of KPC-53 (orange).

DISCUSSION

Here, we characterized a natural variant of KPC-3, the KPC-53 β-lactamase. The peculiarity of this enzyme is the duplication of L167E168 residue in the Ω-loop element. The duplication of these two amino acid residues was described in KPC-25 (GenBank accession no. NG_051167.1), KPC-31 (GenBank accession no. NG_055494.1), and KPC-40 variants (14). KPC-31 has an aspartic acid-to-tyrosine (D179Y) substitution at position 179 in the Ω-loop of KPC-3, which is known to confer resistance to CZA (15). In addition to L167E168 duplication, the KPC-25 and KPC-40 showed Y274H and Y244S substitutions, respectively.

Antimicrobial susceptibility.

Also in our study, CZA association worked better on E. coli XL-1/pBC/KPC-3 than E. coli XL-1/pBC/KPC-53 with MIC reductions of 128- and 4-fold, respectively, showing that KPC-53 was more resistant to avibactam than KPC-3. The MIC results agree with kinetic data obtained for inhibitors. This is particularly true for avibactam, which acted as an inactivator for KPC-3, unlike KPC-53. The MIC values for both KPC-3 and KPC-53 against imipenem and meropenem were low in contrast to the k_cat values determined. Low MICs for carbapenems in carbapenemase-producing E. coli recombinant strains are not unusual (19, 20).

Structural and kinetic analysis.

Comparing the 3D structure of KPC-53 to that of KPC-2/KPC-3, we demonstrated that L167E168 duplication seems to induce the elongation of the short α-helix E168-N170, which in KPC-53 spans from E168 to N172 (N170 in KPC-2 and KPC-3). The Cα backbone of the catalytic residues was found to be invariant with respect to the model templates KPC-2 and KPC-3, although E166 displays in each of the five simulated models for the optimized Ω-loop region, a movement of the backbone carbon. The E166 in KPC-53 is located at the end of the highly ordered region of the new α-helix and the beginning of the highly disordered region of the Ω-loop. The L167E168 duplication in KPC-53 destabilized the hydrogen bonding network inside the Ω-loop structure, resulting in a substantial reduction in the catalytic efficiencies toward most of the tested β-lactams. Moreover, it is plausible that the elongation of the α-helix might decrease the flexibility of the loop during the deacylation step; this effect is much more evident for small substrates as carbapenems. In KPC-53, the greater distance observed between E166 and N170 seems to affect the deacylation step of the β-lactams hydrolysis with a dramatic reduction in k_cat values for most β-lactams. This behavior is remarkable for carbapenems. Indeed, compared to KPC-3, KPC-53 exhibited 2,700- and 2,125-fold decreases in k_cat values for imipenem and meropenem, respectively. Our previous study demonstrated that, in GES variants, substitutions in the Ω-loop (positions 170 and 174) improved catalytic efficiencies against carbapenems (19, 20). In a recent study, Furey et al. characterized KPC-2 mutants deficient for carbapenem hydrolysis and stated that the environment of E166 is important for carbapenemase activity (12). In KPC-53, ceftazidime hydrolysis was 4-fold lower than that of KPC-3, even if their k_cat/K_m values were similar to each other. Amino acid substitutions in the Ω-loop of KPC-2 changed the hydrolysis of extended-spectrum cephalosporins, particularly ceftazidime, enhancing the flexibility of the loop caused by disruption of the R164/D179 salt bridge (21, 22). In “in vitro” inhibition experiments, avibactam, tazobactam, and clavulanic acid acted as transient inactivators for KPC-53 with a low deacylation constant. To investigate structural changes in this variant, tryptophan fluorescence measurements and molecular modeling were carried out. Since the fluorescence of tryptophan is affected by the polarity of its microenvironment, the decrease in the intrinsic fluorescence intensity of KPC-53 with respect to KPC-3 indicates that the L167E168 duplication in the Ω-loop led to some local conformational changes. In particular, W165, adjacent to the duplication in the Ω-loop, could be involved in the modulation of intrinsic fluorescence.

Concluding remarks.

In conclusion, KPC-53 is an enzyme less stable than parental KPC-3, and it has lost the ability to hydrolyze efficiently β-lactams, particularly carbapenems. Furthermore, the L167E168 duplication modifies the interactions between residues involved in the catalytic pocket, changing the flexibility of the Ω-loop, which is directly coupled with the catalytic properties of the KPC enzymes. In class A β-lactamases, the Ω-loop contains residues positioned between R164 and D179 to form the “floor” of the active site of these enzymes. All class A β-lactamases showed similar conformational topology of the Ω-loop, where the N- and C-terminal ends form a typical “bottleneck” (21, 23). The 16 amino acid residues characterizing the Ω-loop form multiple connections to each other: the ionic bond between R164 and D179 or interactions between W165 and L169 (23, 24). In the Ω-loop, E166 and N170 play a crucial role in β-lactam hydrolysis participating in the deacylation step of catalysis.

MATERIALS AND METHODS

Organisms.

The strains examined included E. coli Nova Blue (endA1 hsdR17 [r_K12^– m_K12⁺] supE44 thi-1 recA1 gyrA96 relA1 lac F′ [proA⁺B⁺ lacI^qZΔM15::Tn10]; Tet^r), E. coli BL21(DE3) [B F^– ompT gal dcm lon hsdSB(r_B^– m_B^–) λ(DE3 [lacI lacUV5-T7p07 ind1 sam7 nin5]) [malB⁺]K-12(λS)], and E. coli XL-1 (recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′ proAB lacI^qZΔM15 Tn10; Tet^r]).

Antibiotics.

Meropenem was from AstraZeneca (Milan, Italy). Imipenem and ertapenem were from Merck Sharp & Dohme (Rome, Italy). Nitrocefin was kindly provided by Shariar Mobashery (Notre Dame University, South Bend, IN). Tazobactam was from Wyeth-Lederle (Catania, Italy), and clavulanic acid was from GlaxoSmithKline (Verona, Italy). Avibactam and other antimicrobial agents were purchased MedChemExpress (DBA, Italy) and Sigma-Aldrich (Milan, Italy), respectively. The molar extinction coefficients and the wavelengths of the β-lactams (Δε) used in the assay were as follows: benzylpenicillin (Δε₂₃₅ = −775 M⁻¹ cm⁻¹), carbenicillin (Δε₂₃₅ = –780 M⁻¹ cm⁻¹), cefazolin (Δε₂₆₀ = −7,400 M⁻¹ cm⁻¹), ceftazidime (Δε₂₆₀ = −9,000 M⁻¹ cm⁻¹), nitrocefin (Δε₄₈₂ = 15,000 M⁻¹ cm⁻¹), imipenem (Δε₃₀₀ = −9,000 M⁻¹ cm⁻¹), meropenem (Δε₂₉₇ = −6,500 M⁻¹ cm⁻¹), and ertapenem (Δε₂₉₈ = −7,500 M⁻¹ cm⁻¹).

Cloning of bla_KPC-53.

The bla_KPC-53 gene was amplified from K. pneumoniae LC-1825/18 clinical isolate (16) using primers elsewhere reported (17) and was cloned into pET-24a(+) and pBC-SK vectors. Transformation in E. coli competent cells was performed by using heat shock. The plasmid transfer was confirmed by PCR and automated sequencing (ABI Prism 3500; Life Technologies, Monza, Italy).

Overexpression and purification of KPC-5.

E. coli BL21(DE3) cells carrying the recombinant plasmid pET24-bla_KPC-53 were grown in 2 L of Luria-Bertani (LB) medium with 50 mg/L kanamycin at 37°C in an orbital shaker (180 rpm). The bacteria were grown at 37°C until reaching an optical density at 600 nm (OD₆₀₀) of 0.8. Then, 0.4 mM IPTG (isopropyl-β-d-thiogalactopyranoside) was added, and the culture was incubated at 22°C for 18 h. Cells were harvested by centrifugation at 10,000 × g for 10 min at 4°C and washed twice with 25 mM sodium phosphate buffer (pH 7.0). The crude extract was obtained by sonication on ice (five cycles at 60 W with a 2-min break). The lysate was centrifuged at 100,000 × g for 30 min, and the cleared supernatant was purified, adopting the same procedure used for the KPC-3 enzyme, as reported previously (17). In detail, supernatant was dialyzed overnight at 4°C in 25 mM sodium acetate buffer (pH 5.2) and loaded onto a SP Sepharose FF equilibrated with the same buffer. The column was washed extensively to remove unbound proteins, and the β-lactamase was eluted with a linear gradient of NaCl (0 to 1 M) in the same buffer. Active fractions were pooled, dialyzed in 25 mM sodium phosphate buffer (pH 7.0), and loaded onto a Sephacryl S-100 column (XK16/70; bed volume, 130 mL) equilibrated with 25 mM sodium phosphate buffer (pH 7.0) and 0.15 M NaCl. The active fractions were pooled and stirred at −40°C.

Determination of kinetic parameters.

Kinetic experiments were performed following the hydrolysis of each substrate at 25°C in 25 mM sodium phosphate buffer (pH 7.0). Data were collected with a Perkin-Elmer Lambda 25 spectrophotometer (Perkin-Elmer Italia, Monza, Italy). Steady-state kinetic experiments were determined under initial-rate conditions using the Hanes linearization method (25). Kinetic parameters were determined under initial-rate conditions using the Origin Pro 8.5.1 to generate Michaelis-Menten curves. For K_m values higher than 1 mM, the K_m was determined as K_i using nitrocefin as the reporter substrate (18). Competitive inhibition assays were performed using the following equation: v₀/v_i = 1 + [(K_m × I)/(K_m + S) × K_i], where v_i and v₀ represent the initial rates of hydrolysis of nitrocefin with and without inhibitor, respectively. I is the concentration of inhibitor or poor substrate, K_i is the inhibition constant, K_m is the Michaelis constant, and S is the concentration of the reporter substrate. The plot of v₀/v_i versus I yielded a straight-line slope: K_m/(K_m + S) × K_i (18).

In compounds behaving as transient inhibitors, the accumulation and slow hydrolysis of EC* were studied based on the following model:

$$\text{E}\hspace{0.17em}+\hspace{0.17em}\text{C\hspace{0.17em}}\stackrel{K}{\rightleftarrows }\text{\hspace{0.17em}\hspace{0.17em}EC\hspace{0.17em}}\stackrel{k_{+2}}{\to }\text{EC}*\stackrel{k_{+3}}{\to }\text{E}\hspace{0.17em}+\hspace{0.17em}\text{P}$$

where, E is the enzyme, C is the substrate, EC is the Henri-Michaelis complex, EC* is the acyl-enzyme complex, and P is the hydrolysis product. k₊₂ and k₊₃ are the first-order acylation and deacylation constants, respectively. K is the dissociation constant of the Henry-Michaelis complex.

In the case of poor substrates, the values of ki (first-order rate constant characterizing the EC* accumulation) were obtained by time course hydrolysis of nitrocefin according to the following equations:

$$(v_t\hspace{0.17em}-\hspace{0.17em}{v}_{\text{ss}})/(v_0\hspace{0.17em}-\hspace{0.17em}{v}_{\text{ss}})=e^{–\text{kit}}$$ (1)

where v₀, v_t, and v_ss are the rate transformation of substrate at time zero, t, and steady state, respectively:

$$\mathit{ki}=k_{+3}\hspace{0.17em}+\hspace{0.17em}\frac{k_{+2}[\text{C}]}{[\text{C}]\hspace{0.17em}+\hspace{0.17em}K(1\hspace{0.17em}+\hspace{0.17em}[S]/K_{m}S)}$$ (2)

where [S] is the concentration of reporter substrate and K_mS is the K_m of the reporter substrate, respectively. The condition [S] ≈ K_m, where [S] is the concentration of substrate reporter, was respected. If ki varies linearly with [C] (indicating that the range of [C] = K), the k₊₂/K value is calculated from the slope of the line, and k₊₃ is obtained from the extrapolation at [C] = 0 (17). In the case of avibactam and tazobactam for KPC-3, the plot ki versus [C] was not linear; k₊₃ = 0, k₊₂, and K were calculated by plotting [C]/ki versus [C] (18, 26). Each kinetic value is the mean of three different measurements; the error was below 10%.

Antimicrobial susceptibility.

The MICs were performed by conventional microdilution procedure, as suggested by the Clinical and Laboratory Standard Institute, using a bacterial inoculum of 5 × 10⁵ CFU/mL (27). IPTG was added to the cation-adjusted Mueller-Hinton broth at a concentration of 0.4 mM. The experiments were performed in triplicate using E. coli XL-1/pBC/KPC-53, E. coli XL-1/pBC/KPC-3, and E. coli XL-1/pBC-SK. Tazobactam and avibactam were used at a fixed concentration of 4 μg/mL. Clavulanic acid was used in association with amoxicillin at a ratio of 2:1 (amoxicillin-clavulanic acid).

Thermo Fluor assay.

The thermal stabilities of KPC-53 and KPC-3 were determined using a fluorescence-based thermal stability assay (protein thermal shift kit; Thermo Fisher Scientific, Monza, Italy) in a 7500 Fast real-time PCR system (Applied Biosystems, Monza Italy). The protein melt reaction mix (20 μL total) was added to the 96-well PCR plate. The melt reaction mix included 1 μg of each enzyme, protein shift dye (8×), and 25 mM sodium phosphate buffer (pH 7.0). The plate was heated from 25°C (2 min) to 99°C (2 min) with a heating rate of 1°C/min. The fluorescence intensity was measured with Ex/Em value of 490/530 nm. Analysis of Boltzmann T_m (T_mB) was carried out by using Protein Thermal Shift software, version 1.4. The melting temperature, T_mB, was calculated by fitting data in the region of analysis to the Boltzmann equation (Protein Thermal Shift software).

Fluorescence emission spectra.

Fluorescence studies were carried out on a Perkin-Elmer LS-50B spectrofluorometer. The protein concentrations were 8 μg/mL. The buffer used was 25 mM sodium phosphate (pH 7.0). The excitation wavelength was 280 nm, and the emission spectra, in the range of 300 to 500 nm, were recorded at 25°C.

Molecular modeling.

The 3D structure of KPC-53 was obtained by in silico comparative protein structure modeling using Modeller 10.1 (release 12156; https://salilab.org/modeller/) (28). Briefly, the amino-acidic sequence of the KPC-53 protein was aligned with the known crystal structures KPC-2 (PDB 5MGI) and KPC-3 (PDB 6QWD), yielding a preliminary 3D structure by comparative modeling (29). A subsequent loop region refinement has been performed by generating the five most plausible loop models, whose reliability was evaluated by the statistical potentials (GA341) score, the normalized discrete optimized protein energy (z-DOPE) score, and the RMSD between the coordinates of the backbone carbon atoms in the model and the native structure. Finally, the generated structures were visually inspected using ChimeraX 1.2.5, which was also used for figure generation (30, 31).