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Significant role of Asn-247 and Arg-64 residues in close proximity of the active site in maintaining the catalytic function of CTX-M-15 type β-lactamase

Members of Enterobacteriaceae cause antibiotic-resistant infections worldwide. One such marker, CTX-M-15, expressed by Enterobacteriaceae produces β-lactamases, which hydrolyze the cephalosporin group of antibiotics, such as cefotaxime, used in the treatment of both Gram-positive and negative bacterial infections. Amino acid residues present in close proximity of the active site might also play a major role in the structure and function of CTX-M-15, hence the objective of this study was to investigate the significance of two amino acid residues, Asn-247 and Arg-64, present near to the active site in the hydrolysis of cefotaxime. blaCTX-M-15, cloned from the E. cloacae strain, and using Polymerase Chain Reaction (PCR)-based site-directed mutagenesis, Asn247Val and Arg64Leu mutations were introduced. The minimum inhibitory concentrations of cefotaxime for the CTX-M-15 (N247V) and CTX-M-15 (R64L) mutants were reduced by 512 and 128 fold, respectively. Proteins/enzymes of wild-type CTX-M-15, CTX-M-15 (N247V) and CTX-M-15 (R64L) mutants were expressed and purified. Kinetic studies showed that the catalytic efficiencies of the N247V mutant and R64L mutant enzymes in the hydrolysis of cefotaxime were reduced by 89.66% and 71.11%, respectively. Circular dichroism spectroscopic studies showed considerable changes in the α-helical content of the mutant enzymes. A fluorescence study showed that N247V mutant-cefotaxime and R64L mutant-cefotaxime underwent complex formation with strong interactions. The study provides an understanding of the crucial role of the amino acid residues asparagine 247 and arginine 64 present in close proximity of the active site in the hydrolytic mechanism of CTX-M-15 type β-lactamases. Hence, Asn-247 and Arg-64 can be used as potential target sites for the design of inhibitory molecules against CTX-M-15-producing bacterial strains.


Introduction
Enterobacteriaceae belonging Gram-negative bacteria cause various infections in the human population worldwide, including infections of the respiratory tract, skin, joints, bone, so tissues, urinary tract, central nervous system, eyes, etc. The current issues related to the emergence and spread of resistance to currently available antibiotics in Enterobacteriaceae are making the treatment of serious nosocomial infections a challenge. 1 Emerging resistance in Enterobacteriaceae is thus a serious problem that requires immediate attention. The production of extended spectrum b-lactamases (ESBLs) in members of Enterobacteriaceae is one of the major concerns of infection control due to resistance. In response to environmental pressure, in time new variants are evolving via mutation to gain stability and tness. 2 The production of these resistance conferring enzyme (ESBL) producing Gram-negative bacteria is the reason for higher mortality and morbidity rates in the human population. 3 Moreover, the diseases caused by such bacteria are difficult to identify by routine laboratory assays, leading to delayed diagnosis and therapy. Broad spectrum blactam antibiotics are used for the treatment of both Grampositive and negative bacterial infections, and include penicillins, cephalosporins, monobactams and carbapenems. 4 These antibiotics have a b-lactam ring in their molecular structure. The primary mechanism of the development of resistance to blactam antibiotics is the cleavage of the b-lactam ring by b-lactamase enzymes through the action of hydrolysis. ESBLs belong to class A b-lactamases, which are also dened as plasmidmediated enzymes that play a role in the hydrolysis of oxyimino-cephalosporins. 5 One of the most common ESBLs is CTX-M-types. 6 In the evolution of the resistance mechanism, CTX-M b-lactamases are considered as the base. Genetic incorporation of CTX-M via mobilization into various hierarchical structures and the selective force exerted by cefotaxime (a cephalosporin) has fuelled a mutational event leading to the diversication of various newer clusters of CTX-M and thereby the emergence of several variants in the population. 7 It has been reported that around 20% of K. pneumoniae infections and 31% Enterobacter species infections in ICU departments in the United States are caused by strains that are resistant to third generation cephalosporins. 1 CTX-M stands for cefotaxime hydrolyzing b-lactamase family and hence has the ability to hydrolyze the drug cefotaxime. 8 However, majorly dispersed infections are caused by the CTX-M-15 genotype of the CTX-M family. 7,9 In India, CTX-M-15 has been reported from six unrelated members of Enterobacteriaceae, one K. pneumonia, one E. aerogenes, and four E. coli strains, and is considered as one of the most widespread ESBLs. 10 Its extensive dissemination has led to difficulty in the treatment of the community and hospital acquired infections. 11,12 CTX-M-15 type ESBL plays a major role in the hydrolysis of cefotaxime. 13 There have been several studies on the relevance of active site residues in the catalytic efficiency of CTX-M-15. 13 However, residues present in close proximity of the active site might also play a crucial role in the structural conguration of the CTX-M-15 enzyme in maintaining its hydrolytic efficiency towards antibiotics.
Hence, this study was designed to understand the role of the amino acid residues asparagine 247 and arginine 64 present in close proximity of the active site in the overall activity of CTX-M-15 by replacing them with valine and leucine, respectively, so that if the ndings are signicant, they could be used as potential target sites for the design of appropriate inhibitors.

Antibiotics/chemicals
The antibiotic cefotaxime was purchased from Sigma chemical co. (St. Louis, MO). The substrate nitrocen, which was used in the enzyme kinetic studies, was purchased from Calbiochem (USA). Analytical grade reagents and chemicals were used for the study. Double distilled water was used to carry out all the experiments.

Site-directed mutagenesis and cloning
For the extraction of the plasmid harbouring CTX-M-15 gene from the Enterobacter cloacae strain, a Qiagen plasmid extraction kit was used.
The conditions used for the PCR were denaturation at 95 C for 30 seconds, annealing of the primers at 54 C for 25 seconds and extension at 72 C for 40 seconds. 35 cycles of this process were carried out. 15 The promoter region was not present in the amplied gene, since vector pQE-2 in which we were going to ligate the genes, already has a promoter region. The amplied PCR products (bla CTX-M-15 , bla CTX-M-15(N247V) and bla CTX-M-15(R64L) ) and the high copy cloning vector pQE-2 were double digested using NdeI and HindIII restriction endonucleases. Then in a pQE-2 vector was grown in 100 mg ml À1 of ampicillin containing Luria-Bertani broth. This culture was used to inoculate 100 mg ml À1 of ampicillin containing 1 liter of Luria-Bertani culture and the bacteria were allowed to grow at 120 rpm at 37 C, until 0.6-0.8 optical density was reached at a wavelength of 600 nm. 16 For the expression of the CTX-M-15 protein and the CTX-M-15 (N247V) and CTX-M-15 (R64L) mutant proteins, the respective cultures of each that had reached an absorbance of 0.6-0.8 were cooled and induced using 0.2 mM IPTG (Isopropyl b-D-1-thiogalactopyranoside) as an inducer. The induced cultures were le for 12-16 h at 16 C at 150 rpm. Aer that, the cells were collected by centrifugation and suspended in 20 ml of lysis buffer (containing 50 mM of sodium phosphate at pH 8.0, 300 mM of NaCl and 10 mM of imidazole) and b-mercaptoethanol (0.1%). Then, sonication was performed at 35% amplitude for 10 min, to rupture the cells. By centrifuging at 12 000 rpm for 30 min, the supernatants containing protein were obtained from the rupture cells dissolved in lysis buffer. These supernatants were loaded onto a Ni-NTA column, which was used for the purication of the desired protein. Three Ni-NTA protein purication columns were pre-equilibrated with lysis buffer and the supernatants were then loaded onto it and allowed to bind. The columns were then washed with lysis buffer containing 50 mM of imidazole. To elute bound proteins, phosphate buffer saline (PBS) containing 50 mM of pH 8.0 sodium phosphate and 300 mM of NaCl along with 250 mM of imidazole was used. Aer dialysis in PBS, pure proteins/ enzymes of CTX-M-15, CTX-M-15 (N247V) mutant and CTX-M-15 (R64L) mutant were obtained. Using the 25, 440 M À1 cm À1 molar extinction coefficient, the concentration of the proteins/ enzymes were calculated at 280 nm. 17 Antimicrobial susceptibility testing The minimum inhibitory concentration (MIC) of the antibiotic cefotaxime for wild-type and mutant cells was determined using a microdilution method according to guidelines laid out by the CLSI (Clinical Laboratory Standards Institute). 18 The lowest concentrations of cefotaxime that completely inhibited visible growth of wild and mutant-carrying bacterial cultures were reported as MIC values.

Steady-state enzyme kinetics study
To understand the effect on the CTX-M-15 b-lactamase enzyme upon insertion of the N247V and R64L mutations, an enzyme kinetics study was carried out. For the hydrolytic activities of CTX-M-15, and the CTX-M-15 (N247V) and CTX-M-15 (R64L) mutant enzymes towards a chromogenic cephalosporin b-lactamase substrate, nitrocen was compared in order to understand the effect of the mutation on the hydrolytic efficiency of each enzyme. This was done by examining the rate of appearance and disappearance of nitrocen. 19 The experiment was performed using a spectrophotometer (UV-1800) purchased from Shimadzu International Co. Ltd., Kyoto, Japan. By varying the nitrocen concentration from 0 to 750 mM, the activities of 6 nM of CTX-M-15, and the CTX-M-15 (N247V) and CTX-M-15 (R64L) mutant enzymes were screened. 20 mg ml À1 of BSA was added to 6 nM of the enzymes for dilution and to prevent denaturating prior to screening, since at this concentration of BSA the activities of the enzymes will not be inuenced. 20 Hydrolysis of nitrocen was measured for 70 s at 486 nm and the observed absorbance was used to measure the concentration of nitrocen by taking the molar extinction coefficient 15 000 M À1 cm À1 . 16 The kinetics were measured at 298 K in the presence of pH 7.4 sodium phosphate buffer (50 mM).
Another enzyme kinetics study was carried out to identify the effect of mutations on the hydrolysis of cefotaxime by the CTX-M-15 enzyme. For that, the activities of 6 nM each of CTX-M-15, and the CTX-M-15 (N247V) and CTX-M-15 (R64L) mutant enzymes were monitored at 298 K by varying the concentration of cefotaxime from 0 to 850 mM in the presence of 50 mM of pH 7.4 sodium phosphate buffer. Hydrolysis of cefotaxime was measured for 70 s at 264 nm by taking the value of molar extinction coefficient À7250 M À1 cm À1 . 21 Using the following eqn (1) and (2), the kinetic parameters K m , k cat and k cat /K m were determined:

CD spectroscopic study
To gain insight into the conformation of the secondary structure of the CTX-M-15 enzyme upon insertion of the mutation, CD (circular dichroism) spectroscopy was carried out. The study was performed using a J-810 spectropolarimeter (PTC-423S/15) purchased from Jasco International Co. Ltd., Tokyo, Japan equipped with a water bath and temperature controller (Peltier type). Far UV-CD spectra of 5 mM of CTX-M-15, and the CTX-M-15 (N247V) and CTX-M-15 (R64L) mutant enzymes were recorded in a 0.1 cm cuvette at 298 K. The instrument was calibrated using (+)-10-camphor sulfonic acid and the scan speed and response time were set at 100 nm min À1 and 1 s, respectively. Each measurement was corrected using the appropriate blanks. Using the following eqn (3) (4) and (5), respectively. 23 % a-helix ¼ ½MRE208 nm À 4000 33 000 À 4000 Â 100 Studies based on uorescence spectroscopy (N247V) and CTX-M-15 (R64L) mutant-cefotaxime interactions, another uorescence spectroscopy study was carried out at 298 K for each enzyme. To a 450 ml sample of the enzymes at a concentration of 2 mM, in the presence of sodium phosphate buffer (50 mM, pH 7.4), 2 mM of cefotaxime was added successively until an 18 mM concentration was reached. The samples were excited at 295 nm and in fast scanning mode the uorescence intensities were measured between 300-450 nm wavelengths. 26 The settings of the excitation and emission slits were made at 5 nm. Corrections for the inner lter effect were made for all uorescence intensities. To analyze the uorescence intensity at the emission maxima, the following Stern-Volmer eqn (6) was used: 25 where F and F are the uorescence intensities of CTX-M-15, and the CTX-M-15 (N247V) and CTX-M-15 (R64L) mutant enzymes in the absence and presence of cefotaxime, respectively. The Stern-Volmer constants (K sv ) and bimolecular quenching rate constants (K q ), which are referred to as binding parameters, were calculated using eqn (6) and (7).
[Q] corresponds to the concentration of the quencher cefotaxime and s corresponds to the mean lifetime uorescence of the aromatic amino acid tryptophan present in the CTX-M-15 enzyme in the absence of cefotaxime, which is around 4.31 Â 10 À9 s.
The other binding parameters, K a (binding constant) and n (no. of binding sites), were deduced using the following modi-ed Stern-Volmer eqn (8): 27
The MIC of cefotaxime for the bla CTX-M-15 gene harbouring the DH5a cells was reported to be 512 mg ml À1 , 28 which is in a high resistance range, the MIC of cefotaxime for the bla CTX-M-15(N247V) gene harbouring DH5a cells was found to be 1 mg ml À1 and the MIC for bla CTX-M-15(R64L) harbouring DH5a cells was found to be #4 mg ml À1 , values that are in the sensitive range (Table 1) The major concern of CTX-M-15 carrying bacterial cells is its ability to hydrolyze the antibiotic cefotaxime. So, in order to understand the effect of the mutations on the hydrolytic ability of the CTX-M-15 enzyme to hydrolyze cefotaxime, another enzyme kinetics study was carried out. The Michaelis-Menten plots and the corresponding Lineweaver-Burk plots are shown in Fig. 2. The Lineweaver-Burk plots were used to deduce the kinetic parameters (Tables 2 and 3 (Table 4). When the spectrum of the wild CTX-M-15 was recorded, the MRE values were found to be À14 196.3 and À14 237.2 deg cm 2 dmol À1 at 208 and 222 nm, respectively, and the percentages of the a-helical content present at 208 and 222 nm were 35.16% and 39.26%, respectively. When the spectrum of the CTX-M-15 (N247V) mutant was recorded, the MRE values had changed to À16 473.2 and À14 681.5 deg cm 2 dmol À1 at 208 and 222 nm, respectively and the percentages of the a-helical content present at 208 and 222 nm had changed to 43.01% and 40.73%,    To monitor the effect of the mutations on the uorescence intensity of the CTX-M-15 enzyme, uorescence spectra were recorded. The uorescence spectra of the CTX-M-15, CTX-M-15 (N247V), and CTX-M-15 (R64L) were recorded at 298 K (Fig. 4). A considerable decrease in the uorescence intensity curves was observed in the case of the mutant enzymes. Moreover, since the study was focused on the effect of the mutation on the  cefotaxime binding and its hydrolysis, to understand the interaction of the antibiotic cefotaxime with CTX-M-15, CTX-M-15 (N247V) and CTX-M-15 (R64L), uorescence spectroscopy was carried out. A progressive decrease in the uorescence intensity curves of the wild enzyme and mutant enzymes was observed at 298 K due to a quenching phenomenon (Fig. 5). Using the Stern-Volmer eqn (6) and (7) and modied Stern-Volmer eqn (8), the Stern-Volmer and modied Stern-Volmer plots were determined for CTX-M-15 and the CTX-M-15 (N247V) mutant enzyme (Fig. 6) and for CTX-M-15 and the CTX-M-15 (R64L) mutant enzyme (Fig. 7). Also using eqn (6)-(8), binding parameters were calculated ( Table 5). The K sv (Stern-Volmer constant) values were found to be in the order of 10 4 M À1 . The

Discussion
The spread of CTX-M-15 and its variants is one of the major concerns in infection control, caused by multi-drug resistant strains. 30 Due to continuous evolution, newer variants of CTX-M-15 are continuously arising, thus making the situation much worse. Several studies have shown the signicance of active site residues of CTX-M-15, such as Ser-70, Thr-235, Ser-130, Asn-104, Arg-274, Ser-237, etc., in substrate binding and catalysis. 13,16 However, other residues near to the active site might also contribute signicantly in determining the broad specicity of the CTX-M-15 enzyme, as observed aer closely examining the CTX-M-15 structure. Also, in the course of CTX-M-15 variant evolution, the residues present near to the active site play a major role. Therefore, a detailed study is needed to be carried out to understand the importance of these residues in the structure and function of the CTX-M-15 enzyme that lead to evolution. In this study, we explored the signicance of two residues, Asn-247 and Arg-64, present near to the active site, in the structure and function of CTX-M-15 (PDB-ID: 5T66) by replacing them with Val-247 and Leu-64, respectively. Asn-247 and Arg-64 are located in close proximity of the active site at 9.7Å and 18.8Å distance from the active site, respectively, as shown in Fig. 8.
To evaluate the effect of the mutation on the resistance pattern, the MICs of E. coli   fold. Similarly, on replacing Arg-64 with Leu-64 in bla CTX-M-15 , the MIC of cefotaxime was reduced by 128 fold. Using puried proteins/enzymes, steady-state enzyme kinetics studies were performed. First of all, the enzyme kinetics were investigated by varying the concentration of nitrocen to monitor the effect on the enzyme upon mutation. Nitrocen is a chromogenic cephalosporin substrate, which exhibits a hydrolytic prole of a characteristic b-lactamase enzyme. The b-lactam ring of nitrocen is hydrolyzed by a blactamase enzyme, producing a shi in the UV absorption, which is detected as a measure of the b-lactamase activity. The K m value of the N247V mutant was increased considerably, which means that the affinity of the N247V mutant towards the substrate was reduced considerably. Similarly, the K m value of the R64L mutant was increased much more than that of the N247V mutant, indicating much more of a decrease in the affinity of the R64L mutant towards the substrate. The overall catalytic efficiency of the CTX-M-15 enzyme on changing Asn-247 to Val-247 towards the hydrolysis of nitrocen was decreased by 69.01%. And, the efficiency of the enzyme on changing Arg-64 to Leu-64 towards the hydrolysis of nitrocen was decreased by 94.39%. CTX-M-15 is characterized by its ability to hydrolyze the antibiotic cefotaxime. So, to gain an insight into the effect of the mutation on the cefotaxime hydrolytic ability of the enzyme, another enzyme kinetics study was carried out. An increase in K m and a signicant decrease in k cat was observed for the N247V mutant enzyme suggested a decrease in the affinity of the enzyme towards cefotaxime and a decrease in the catalytic activity of the enzyme towards the hydrolysis of cefotaxime. The catalytic efficiency (k cat /K m ) of the N247V mutant enzyme towards the hydrolysis of cefotaxime was decreased by 89.66%. Similarly, a signicant decrease in the K m and k cat values was observed for the R64L mutant enzyme, suggesting an increase in the affinity and a decrease in the catalytic activity of the enzyme. Overall, the catalytic efficiency (k cat /K m ) of the R64L mutant enzyme towards the hydrolysis of cefotaxime was decreased by 71.11%.
The kinetics data, together with the MIC values, clearly showed that the Asn247Val and Arg64Leu mutants reduced the ability of the CTX-M-15 enzyme to hydrolyze cefotaxime. CD studies help to determine the secondary and tertiary structures of the protein, as well as to determine intermediate states, such as molten globules, during the alteration in the conformation of a particular protein/enzyme. 31 The fraction of protein/enzyme molecules present in various conformations of the secondary structure, such as a-helices, b-sheets, beta-turn, random coil, etc., can also be estimated. Hence, to investigate the effect of the mutation on the overall structure and stability of the CTX-M-15 enzyme, CD spectroscopic studies were performed. The CD spectrum of wild CTX-M-15 displayed a characteristic a-helical protein curve with minima at 208 nm and 222 nm, resembling the previously reported far UV-CD spectrum of CTX-M-1. 32 On mutating Asn-247 to Val-247 in CTX-M-15, a distorted far-UV CD spectrum was observed with increased a-helical content and MRE values at 208 and 222 nm. On mutating Arg-64 to Leu-64 in CTX-M-15, an altered far-UV CD spectrum was observed with increased a-helical content and a MRE value at 208 nm and decreased a-helical content and MRE value at 222 nm.
Fluorescence spectroscopic studies are carried out to understand the interaction of a particular drug with protein molecules. When an electron from a higher energy level returns back to a lower energy level, uorescence is observed, referred to as photon emission. 33 Fluorescence phenomena involve the absorption of energy in energy levels of the electron and emission at a longer wavelength. Fluorescence spectroscopic studies measure the decrease in the uorescence intensity upon drug binding, known as quenching phenomena. Quenching is either static or dynamic, with static quenching forming the groundstate complex and in dynamic quenching, the quencher interacts with the molecules indirectly. Quenching is observed due to the transfer of energy or due to molecular collisions, rearrangement and interaction in the excited state. 33 This technique also helps in the determination of the conformational

Conclusion
It can be concluded that Asn-247 and Arg-64 play a signicant role in maintaining the overall stability and proper orientation of the active site of the CTX-M-15 enzyme. Hence, these amino acid residues could be targeted for the development of novel inhibitors in the future. The study shows that the non-active site amino acid residues play a very crucial role in modulating the sensitivity of the active site and maintaining the structure and function of the b-lactamase enzyme. The development of inhibitors against them can also be an approach to ght infections against b-lactamase-producing strains.

Funding
The work was supported by the Department of Biotechnology, Government of India, grant no. BT/PR8281/BID/7/448/2013.

Author contributions
LM performed all of the experiments, did the calculations and graph plotting and wrote the manuscript; SK helped in the various experiments, AA helped in the protein purication, AUK designed the study, provided guidance and checked the manuscript.

Conflicts of interest
There are no conicts to declare.

CD Circular dichroism MIC
Minimum inhibitory concentration