Acylation Rate Research Articles

Molecular Basis of Influence of A501X Mutations in Penicillin-Binding Protein 2 of Neisseria gonorrhoeae Strain 35/02 on Ceftriaxone Resistance.

The increase in the resistance of mutant strains of Neisseria gonorrhoeae to the antibiotic ceftriaxone is pronounced in the decrease in the second-order acylation rate constant, k2/KS, by penicillin-binding protein 2 (PBP2). These changes can be caused by both the decrease in the acylation rate constant, k2, and the weakening of the binding affinity, i.e., an increase in the substrate constant, KS. A501X mutations in PBP2 affect second-order acylation rate constants. The PBP2A501V variant exhibits a higher k2/KS value, whereas for PBP2A501R and PBP2A501P variants, these values are lower. We performed molecular dynamic simulations with both classical and QM/MM potentials to model both acylation energy profiles and conformational dynamics of four PBP2 variants to explain the origin of k2/KS changes. The acylation reaction occurs in two elementary steps, specifically, a nucleophilic attack by the oxygen atom of the Ser310 residue and C-N bond cleavage in the β-lactam ring accompanied by the elimination of the leaving group of ceftriaxone. The energy barrier of the first step increases for PBP2 variants with a decrease in the observed k2/KS value. Submicrosecond classic molecular dynamic trajectories with subsequent cluster analysis reveal that the conformation of the β3-β4 loop switches from open to closed and its flexibility decreases for PBP2 variants with a lower k2/KS value. Thus, the experimentally observed decrease in the k2/KS in A501X variants of PBP2 occurs due to both the decrease in the acylation rate constant, k2, and the increase in KS.

Prediction of deleterious non-synonymous SNPs of TMPRSS2 protein combined with Molecular Dynamics Simulations and free energy analysis to identify the potential peptide substrates against SARS-CoV-2.

Globally the SARS-CoV-2 viral infection demands for the new drugs, the TMPRSS2 target plays a vital role in facilitating the virus entry. The aim of the present study is to identify the potential peptide substrate from the Anti-viral database against TMPRSS2 of SARS-CoV-2. The compound screening and variation analysis were performed using molecular docking analysis and online tools such as PROVEAN and SNAP2 server, respectively. The re-docked crystal structure peptide substrate exhibits -128.151 kcal/mol whereas the RRKK peptide substrate shows -134.158 kcal/mol. Further, the selected compounds were proceeded with Molecular Dynamics Simulation, it explores the stability of the complex by revealing the hotspot residues (His296 and Ser441) were active for nucleophilic attack against TMPRSS2. The average Binding Free Energy values computed through MM/GBSA for RRKK, Camostat, and Crystal Structure were shown -69.9278 kcal/mol, -64.5983 kcal/mol, and -63.9755 kcal/mol, respectively against TMPRSS2. The 'rate of acylation' emerges as an indicator for RRKK's efficacy, it maintains the distance of 3.2 Å with Ser441 resembles, whilst its -NH backbone stabilizes at 2.5 Å 'Michaelis Complex' which leads to prevent the entry of SARS-CoV-2 to human cells. The sequence variation analysis explores that the V160 and G6 substitutions are essential to emphasize the uncover possibilities for the ongoing drug discovery research. Therefore, the identified peptide substrate found to be potent against SARS-CoV-2 and these results will be valuable for ongoing drug discovery research.Communicated by Ramaswamy H. Sarma.

Structural and kinetic analysis of the monofunctional Staphylococcus aureus PBP1

Staphylococcus aureus, an ESKAPE pathogen, is a major clinical concern due to its pathogenicity and manifold antimicrobial resistance mechanisms. The commonly used β-lactam antibiotics target bacterial penicillin-binding proteins (PBPs) and inhibit crosslinking of peptidoglycan strands that comprise the bacterial cell wall mesh, initiating a cascade of effects leading to bacterial cell death. S. aureus PBP1 is involved in synthesis of the bacterial cell wall during division and its presence is essential for survival of both antibiotic susceptible and resistant S. aureus strains. Here, we present X-ray crystallographic data for S. aureus PBP1 in its apo form as well as acyl-enzyme structures with distinct classes of β-lactam antibiotics representing the penicillins, carbapenems, and cephalosporins, respectively: oxacillin, ertapenem and cephalexin. Our structural data suggest that the PBP1 active site is readily accessible for substrate, with little conformational change in key structural elements required for its covalent acylation of β-lactam inhibitors. Stopped-flow kinetic analysis and gel-based competition assays support the structural observations, with even the weakest performing β-lactams still having comparatively high acylation rates and affinities for PBP1. Our structural and kinetic analysis sheds insight into the ligand–PBP interactions that drive antibiotic efficacy against these historically useful antimicrobial targets and expands on current knowledge for future drug design and treatment of S. aureus infections.

Esterification of black bean anthocyanins with unsaturated oleic acid, and application characteristics of the product

Esterification of anthocyanins with saturated fatty acids have been widely investigated, while that with unsaturated fatty acids is little understood. In this study, crude extract (purity ∼ 35 %) of cyanidin-3-O-glucoside (C3G) from black bean seed coat was utilized as reaction substrate, and enzymatically acylated with unsaturated fatty acid (oleic acid). Optimization of various reaction parameters finally resulted in the highest acylation rate of 54.3 %. HPLC-MS/MS and NMR analyses elucidated the structure of cyanidin-3-O-glucoside-oleic acid ester (C3G-OA) to be cyanidin-3-O-(6″-octadecene)-glucoside. Introduction of oleic acid into C3G improved the lipophilicity, antioxidant ability, and antibacterial activity. Further, the color and substance stability analyses showed that the susceptibility of C3G and C3G-OA to different thermal, peroxidative, and illuminant treatments were highly pH dependent, which suggested individual application guidelines. Moreover, C3G-OA showed lower toxicity to normal cell (QSG-7701) and better inhibitory effect on the proliferation of HepG2 cells than C3G, which indicated its potential anti-tumor bioactivity.

Peptide Acylation in Aliphatic Polyesters: a Review of Mechanisms and Inhibition Strategies.

Biodegradable polyesters are widely employed in the development of controlled release systems for peptide drugs. However, one of the challenges in developing a polyester-based delivery system for peptides is the acylation reaction between peptides and polymers. Peptide acylation is an important factor that affects formulation stability and can occur during storage, in vitro release, and after drug administration. This review focuses on the mechanisms and parameters that influence the rate of peptide acylation within polyesters. Furthermore, it discusses reported strategies to minimize the acylation reaction.

Ureidopenicillins Are Potent Inhibitors of Penicillin-Binding Protein 2 from Multidrug-Resistant Neisseria gonorrhoeae H041.

Effective treatment of gonorrhea is threatened by the increasing prevalence of Neisseria gonorrhoeae strains resistant to the extended-spectrum cephalosporins (ESCs). Recently, we demonstrated the promise of the third-generation cephalosporin cefoperazone as an antigonococcal agent due to its rapid second-order rate of acylation against penicillin-binding protein 2 (PBP2) from the ESC-resistant strain H041 and robust antimicrobial activity against H041. Noting the presence of a ureido moiety in cefoperazone, we evaluated a subset of structurally similar ureido β-lactams, including piperacillin, azlocillin, and mezlocillin, for activity against PBP2 from H041 using biochemical and structural analyses. We found that the ureidopenicillin piperacillin has a second-order rate of acylation against PBP2 that is 12-fold higher than cefoperazone and 85-fold higher than ceftriaxone and a lower MIC against H041 than ceftriaxone. Surprisingly, the affinity of ureidopenicillins for PBP2 is minimal, indicating that their inhibitory potency is due to a higher rate of the acylation step of the reaction compared to cephalosporins. Enhanced acylation results from the combination of a penam scaffold with a 2,3-dioxopiperazine-containing R1 group. Crystal structures show that the ureido β-lactams overcome the effects of resistance mutations present in PBP2 from H041 by eliciting conformational changes that are hindered when PBP2 interacts with the weaker inhibitor ceftriaxone. Overall, our results support the potential of piperacillin as a treatment for gonorrhea and provide a framework for the future design of β-lactams with improved activity against ESC-resistant N. gonorrhoeae.

Development of lipopeptide surfactants from silk sericin and evaluation of their surface active properties

The synthesis of lipopeptides from silk sericin was realized in two steps: enzymatic hydrolysis of protein with Alcalase enzyme and the grafting of lipid chain (C10, C12 or C14) following the Schotten-Baumann reaction. The influence of experimental conditions on the degree of hydrolysis (DH) and the acylation rate (AR) was investigated. Then, equilibrium surface adsorption properties of lipopeptides at air/water interface were studied. The effect of lipopeptide structure, in particular the size of peptide and of the hydrophobic chain, was characterized. Lipopeptides obtained with DH from 20 % to 45 % and AR from 27 % to 41 % were successfully produced. The obtained results demonstrated that sericin peptides have poor amphiphilic properties, whereas lipopeptides efficiently decreased air/water surface tension up to 30.9 mN/m. The surface activity of lipopeptides seem to be closely related to their structure. Sericin lipopeptides show surface properties comparable to those of commercial surfactants, which emphasize the interest of these biomass-derived amphiphilic molecules for potential replacement of petroleum-based surfactants.

Enzymatic acylation of blueberry (Vaccinium ashei) pomace anthocyanins and determination of their cytotoxic properties

To expand the application of acylated blueberry pomace anthocyanins in food and nutraceuticals, their enzymatic acylation reaction and the cytotoxic response were explored. In this study, enzymatic acylation was carried out with cyanidin-3-O-glucoside (C3G) as the main body and Novozyme 435 as the biocatalyst. With a series of improvements, the reaction was carried out under the conditions of lauric acid as the acyl donor, tert-amyl alcohol as the reaction medium, the molar ratio of C3G to lauric acid of 1:10, the reaction time of 24 h and the reaction temperature of 60 °C, and 35.29 % acylation rate was obtained. Moreover, it was found that the lipophilicity, thermostability and photostability of the enzymatic acylated blueberry pomace anthocyanins were improved compared with the untreated blueberry pomace anthocyanins. Cytotoxicity studies showed that acylated blueberry pomace anthocyanins inhibited tumor cells (HeLa, HepG2, and B16 cells) significantly and were less toxic to normal cells (L929 cells) at the maximum concentration (1000 µg/mL) and the longest duration of action (72 h). This study established a method for acylation of blueberry pomace anthocyanins that may be applied to other natural sources of anthocyanins.

The Effectiveness of Imipenem-Relebactam against Ceftazidime-Avibactam Resistant Variants of the KPC-2 β-Lactamase.

Ceftazidime-avibactam was approved by the FDA to treat infections caused by Enterobacterales carrying blaKPC-2. However, variants of KPC-2 with amino acid substitutions at position 179 have emerged and confer resistance to ceftazidime-avibactam. The activity of imipenem-relebactam was assessed against a panel of 19 KPC-2 D179 variants. KPC-2 and the D179N and D179Y variants were purified for biochemical analyses. Molecular models were constructed with imipenem to assess differences in kinetic profiles. All strains were susceptible to imipenem-relebactam, but resistant to ceftazidime (19/19) and ceftazidime-avibactam (18/19). KPC-2 and the D179N variant hydrolyzed imipenem, but the D179N variant's rate was much slower. The D179Y variant was unable to turnover imipenem. All three β-lactamases hydrolyzed ceftazidime at varying rates. The acylation rate of relebactam for the D179N variant was ~2.5× lower than KPC-2. Poor catalytic turnover by the D179Y variant precluded the determination of inhibitory kinetic parameters. Acyl-complexes with imipenem and ceftazidime were less prevalent with the D179N variant compared to the D179Y variant, supporting the kinetic observations that the D179Y variant was not as active as the D179N variant. Relebactam was slower to form an acyl-complex with the D179Y variant compared to avibactam. The D179Y model with imipenem revealed that the catalytic water molecule was shifted, and the carbonyl of imipenem was not within the oxyanion hole. Conversely in the D179N model, imipenem was oriented favorably for deacylation. Imipenem-relebactam overcame the resistance of the D179 variants, suggesting that this combination will be active against clinical isolates harboring these derivatives of KPC-2.

Ultrasonic assisted extraction of anthocyanins from rose flower petal in DES system and enzymatic acylation

Herein, the anthocyanins from rose were extracted, purified and identified by ultrasound-assisted deep eutectic solvents (DES), the resin, and ESI-Q-TOF-MS, respectively. The purified anthocyanins were modified through acylation by means of enzymatic catalysis method to improve the stability of anthocyanins. Compared with traditional extraction agents (ethanol), anthocyanins extraction rate was significantly improved when choline chloride/lactic acid was chosen as extraction solvent, and reached 8.265 mg g−1. The ESI-Q-TOF-MS detection results showed that the main component of the anthocyanins was peonidin-3,5-di-O-glucoside with 65.93% content. Meanwhile, the maximal acylation rate was 95.13% under optimal conditions (0.25 g of anthocyanin, 2.5 mL of pyridine, 4 mL of methyl benzoate; 400 mg of Novocain 435 lipase addition; a water bath at 50 °C under negative pressure of 0.09 Mpa for 9 h). Furthermore, the loss rate of acylated anthocyanins was significantly lower than that of natural anthocyanins under the same temperature or light conditions, and the acylation of anthocyanin could significantly improve stability against temperature, light and pH. The acylated anthocyanin exhibited excellent free radical scavenging activities for DPPH and ABTS+, and IC50 values (4.451 μg mL−1 for DPPH and 7.760 μg mL−1 for ABTS+) displayed its high antioxidant activity.

Stereoselective Synthesis of γ‐(Acyloxy)carboxylic Acids and γ‐Lactones Featuring the Switch of Stereopreference of Candida antarctica Lipase B in Sodium γ‐Hydroxycarboxylate Homologues

AbstractScalable protocols of straightforward synthesis of enantiomeric γ‐(acyloxy)carboxylic acids and γ‐lactones are presented. The key step is lipase‐catalyzed stereoselective acylation of γ‐hydroxycarboxylic acid sodium salt in organic solvent followed by acidification of the product, extraction and acidic relactonization of the unreacted enantiomer. The mixture of γ‐(acyloxy)carboxylic acid and γ‐lactone is separated either by extraction with solution of sodium bicarbonate or by distillation. A switch of enantioinduction of Candida antarctica lipase B along homologous nucleophiles from R configuration of γ‐hydroxyhexanoic acid salt to S configuration of the C7 and longer‐chain homologues has been disclosed. Both enantiomers of γ‐(acyloxy)pentanoic acids; γ‐(acetyloxy)octanoic and ‐nonanoic acids with S configuration; [(1S,5R)‐5‐(chloroacetyloxy)cyclopent‐2‐en‐1‐yl]acetic acid and enantiomeric γ‐lactones derived from them were prepared with e. r. >98.5/1.5. The rates of acylation of C5 to C9 homologous salts differ by three orders of magnitude but remain applicable for preparative synthesis by variation of the enzyme loading and reaction time.

Evolution of Ceftriaxone Resistance of Penicillin-Binding Proteins 2 Revealed by Molecular Modeling.

Penicillin-binding proteins 2 (PBP2) are critically important enzymes in the formation of the bacterial cell wall. Inhibition of PBP2 is utilized in the treatment of various diseases, including gonorrhea. Ceftriaxone is the only drug used to treat gonorrhea currently, and recent growth in PBP2 resistance to this antibiotic is a serious threat to human health. Our study reveals mechanistic aspects of the inhibition reaction of PBP2 from the wild-type FA19 strain and mutant 35/02 and H041 strains of Neisseria Gonorrhoeae by ceftriaxone. QM(PBE0-D3/6-31G**)/MM MD simulations show that the reaction mechanism for the wild-type PBP2 consists of three elementary steps including nucleophilic attack, C-N bond cleavage in the β-lactam ring and elimination of the leaving group in ceftriaxone. In PBP2 from the mutant strains, the second and third steps occur simultaneously. For all considered systems, the acylation rate is determined by the energy barrier of the first step that increases in the order of PBP2 from FA19, 35/02 and H041 strains. Dynamic behavior of ES complexes is analyzed using geometry and electron density features including Fukui electrophilicity index and Laplacian of electron density maps. It reveals that more efficient activation of the carbonyl group of the antibiotic leads to the lower energy barrier of nucleophilic attack and larger stabilization of the first reaction intermediate. Dynamical network analysis of MD trajectories explains the differences in ceftriaxone binding affinity: in PBP2 from the wild-type strain, the β3-β4 loop conformation facilitates substrate binding, whereas in PBP2 from the mutant strains, it exists in the conformation that is unfavorable for complex formation. Thus, we clarify that the experimentally observed decrease in the second-order rate constant of acylation (k2/KS) in PBP2 from the mutant strains is due to both a decrease in the acylation rate constant k2 and an increase in the dissociation constant KS.

The l,d-Transpeptidase LdtAb from Acinetobacter baumannii Is Poorly Inhibited by Carbapenems and Has a Unique Structural Architecture.

l,d-Transpeptidases (LDTs) are enzymes that catalyze reactions essential for biogenesis of the bacterial cell wall, including formation of 3-3 cross-linked peptidoglycan. Unlike the historically well-known bacterial transpeptidases, the penicillin-binding proteins (PBPs), LDTs are resistant to inhibition by the majority of β-lactam antibiotics, with the exception of carbapenems and penems, allowing bacteria to survive in the presence of these drugs. Here we report characterization of LdtAb from the clinically important pathogen, Acinetobacter baumannii. We show that A.baumannii survives inactivation of LdtAb alone or in combination with PBP1b or PBP2, while simultaneous inactivation of LdtAb and PBP1a is lethal. Minimal inhibitory concentrations (MICs) of all 13 β-lactam antibiotics tested decreased 2- to 8-fold for the LdtAb deletion mutant, while further decreases were seen for both double mutants, with the largest, synergistic effect observed for the LdtAb + PBP2 deletion mutant. Mass spectrometry experiments showed that LdtAb forms complexes in vitro only with carbapenems. However, the acylation rate of these antibiotics is very slow, with the reaction taking longer than four hours to complete. Our X-ray crystallographic studies revealed that LdtAb has a unique structural architecture and is the only known LDT to have two different peptidoglycan-binding domains.

Assessing the Potency of β-Lactamase Inhibitors with Diverse Inactivation Mechanisms against the PenA1 Carbapenemase from Burkholderia multivorans.

Burkholderia cepacia complex (Bcc) poses a serious health threat to people with cystic fibrosis or compromised immune systems. Infections often arise from Bcc strains, which are highly resistant to many classes of antibiotics, including β-lactams. β-Lactam resistance in Bcc is conferred largely via PenA-like β-lactamases. Avibactam was previously shown to be a potent inactivator of PenA1. Here, we examined the inactivation mechanism of PenA1, a class A serine carbapenemase from Burkholderia multivorans using β-lactamase inhibitors (β-lactam-, diazabicyclooctane-, and boronate-based) with diverse mechanisms of action. In whole cell based assays, avibactam, relebactam, enmetazobactam, and vaborbactam restored susceptibility to piperacillin against PenA1 expressed in Escherichia coli. The rank order of potency of inactivation in vitro based on kinact/KI or k2/K values (range: 3.4 × 102 to 2 × 106 M-1 s-1) against PenA1 was avibactam > enmetazobactam > tazobactam > relebactam > clavulanic acid > vaborbactam. The contribution of selected amino acids (S70, K73, S130, E166, N170, R220, K234, T237, and D276) in PenA1 toward inactivation was evaluated using site-directed mutagenesis. The S130A, R220A, and K234A variants of PenA1 were less susceptible to inactivation by avibactam. The R220A variant was purified and assessed via steady-state inhibition kinetics and found to possess increased Ki-app values and decreased kinact/KI or k2/K values against all tested inhibitors compared to PenA1. Avibactam was the most affected by the alanine replacement at 220 with a nearly 400-fold decreased acylation rate. The X-ray crystal structure of the R220A variant was solved and revealed loss of the hydrogen bonding network between residues 237 and 276 leaving a void in the active site that was occupied instead by water molecules. Michaelis-Menten complexes were generated to elucidate the molecular contributions of the poorer in vitro inhibition profile of vaborbactam against PenA1 (k2/K, 3.4 × 102 M-1 s-1) and was compared to KPC-2, a class A carbapenemase that is robustly inhibited by vaborbactam. The active site of PenA1 is larger than that of KPC-2, which impacted the ability of vaborbactam to form favorable interactions, and as a result the carboxylate of vaborbactam was drawn toward K234/T235 in PenA1 displacing the boronic acid from approaching the nucleophilic S70. Moreover, in PenA1, the tyrosine at position 105 compared to tryptophan in KPC-2, was more flexible rotating more than 90°, and as a result PenA1's Y105 competed for binding with the cyclic boronate vs the thiophene moiety of vaborbactam, further precluding inhibition of PenA1 by vaborbactam. Given the 400-fold decreased k2/K for the R220A variant compared to PenA1, acyl-enzyme complexes were generated via molecular modeling and compared to the PenA1-avibactam crystal structure. The water molecules occupying the active site of the R220A variant are unable to stabilize the T237 and D276 region of the active site altering the ability of avibactam to form favorable interactions compared to PenA1. The former likely impacts the ability of all inhibitors to effectively acylate this variant enzyme. Based on the summation of all evidence herein, the utility of these newer β-lactamase inhibitors (i.e., relebactam, enmetazobactam, avibactam, and vaborbactam) in combination with a β-lactam against B. multivorans producing PenA1 and the R220A variant is promising.

Size-Driven Inversion of Selectivity in Esterification Reactions: Secondary Beat Primary Alcohols.

Relative rates for the Lewis base-mediated acylation of secondary and primary alcohols carrying large aromatic side chains with anhydrides differing in size and electronic structure have been measured. While primary alcohols react faster than secondary ones in transformations with monosubstituted benzoic anhydride derivatives, relative reactivities are inverted in reactions with sterically biased 1-naphthyl anhydrides. Further analysis of reaction rates shows that increasing substrate size leads to an actual acceleration of the acylation process, the effect being larger for secondary as compared to primary alcohols. Computational results indicate that acylation rates are guided by noncovalent interactions (NCIs) between the catalyst ring system and the DED substituents in the alcohol and anhydride reactants. Thereby stronger NCIs are formed for secondary alcohols than for primary alcohols.

Molecular Features of Cephalosporins Important for Activity against Antimicrobial-Resistant Neisseria gonorrhoeae.

The increasing prevalence of Neisseria gonorrhoeae strains exhibiting decreased susceptibility to extended-spectrum cephalosporins (ESCs) presents a challenge for the successful treatment of gonorrhea infections. To address this challenge, we evaluated a panel of 23 cephalosporins against penicillin-binding protein 2 (PBP2) from the ESC-resistant (ESCR) N. gonorrhoeae strain H041 to determine which molecular features are important for antimicrobial activity. Structure-activity relationships (SARs) developed from acylation rate constants against PBP2 and antimicrobial susceptibilities against the H041 strain of N. gonorrhoeae, and interpreted against docking models, reveal that cephalosporins possessing large, lipophilic R1 side chains and electronegative R2 side chains with planar groups are associated with higher acylation rates against PBP2, but also that these same amphipathic features can lower antimicrobial activity. Based on these studies, we tested cefoperazone, one of the most effective ESCs for targeting PBP2, in the female mouse model infected with H041 and showed that it was equally or more effective than ceftriaxone or gentamicin for clearing infections. Taken together, our results reveal that two U.S. Food and Drug Administration (FDA)-approved agents (cefoperazone, ceftaroline) and one FDA-qualified infectious disease product (ceftobiprole) have potential as first-line treatments for gonorrhea and provide a framework for the future design of cephalosporins with improved activity against ESC-resistant N. gonorrhoeae.

KPC-2 β-lactamase enables carbapenem antibiotic resistance through fast deacylation of the covalent intermediate

Serine active-site β-lactamases hydrolyze β-lactam antibiotics through the formation of a covalent acyl-enzyme intermediate followed by deacylation via an activated water molecule. Carbapenem antibiotics are poorly hydrolyzed by most β-lactamases owing to slow hydrolysis of the acyl-enzyme intermediate. However, the emergence of the KPC-2 carbapenemase has resulted in widespread resistance to these drugs, suggesting it operates more efficiently. Here, we investigated the unusual features of KPC-2 that enable this resistance. We show that KPC-2 has a 20,000-fold increased deacylation rate compared with the common TEM-1 β-lactamase. Furthermore, kinetic analysis of active site alanine mutants indicates that carbapenem hydrolysis is a concerted effort involving multiple residues. Substitution of Asn170 greatly decreases the deacylation rate, but this residue is conserved in both KPC-2 and non-carbapenemase β-lactamases, suggesting it promotes carbapenem hydrolysis only in the context of KPC-2. X-ray structure determination of the N170A enzyme in complex with hydrolyzed imipenem suggests Asn170 may prevent the inactivation of the deacylating water by the 6α-hydroxyethyl substituent of carbapenems. In addition, the Thr235 residue, which interacts with the C3 carboxylate of carbapenems, also contributes strongly to the deacylation reaction. In contrast, mutation of the Arg220 and Thr237 residues decreases the acylation rate and, paradoxically, improves binding affinity for carbapenems. Thus, the role of these residues may be ground state destabilization of the enzyme-substrate complex or, alternatively, to ensure proper alignment of the substrate with key catalytic residues to facilitate acylation. These findings suggest modifications of the carbapenem scaffold to avoid hydrolysis by KPC-2 β-lactamase.

A drug-resistant β-lactamase variant changes the conformation of its active-site proton shuttle to alter substrate specificity and inhibitor potency

Lys234 is one of the residues present in class A β-lactamases that is under selective pressure due to antibiotic use. Located adjacent to proton shuttle residue Ser130, it is suggested to play a role in proton transfer during catalysis of the antibiotics. The mechanism underpinning how substitutions in this position modulate inhibitor efficiency and substrate specificity leading to drug resistance is unclear. The K234R substitution identified in several inhibitor-resistant β-lactamase variants is associated with decreased potency of the inhibitor clavulanic acid, which is used in combination with amoxicillin to overcome β-lactamase-mediated antibiotic resistance. Here we show that for CTX-M-14 β-lactamase, whereas Lys234 is required for hydrolysis of cephalosporins such as cefotaxime, either lysine or arginine is sufficient for hydrolysis of ampicillin. Further, by determining the acylation and deacylation rates for cefotaxime hydrolysis, we show that both rates are fast, and neither is rate-limiting. The K234R substitution causes a 1500-fold decrease in the cefotaxime acylation rate but a 5-fold increase in kcat for ampicillin, suggesting that the K234R enzyme is a good penicillinase but a poor cephalosporinase due to slow acylation. Structural results suggest that the slow acylation by the K234R enzyme is due to a conformational change in Ser130, and this change also leads to decreased inhibition potency of clavulanic acid. Because other inhibitor resistance mutations also act through changes at Ser130 and such changes drastically reduce cephalosporin but not penicillin hydrolysis, we suggest that clavulanic acid paired with an oxyimino-cephalosporin rather than penicillin would impede the evolution of resistance.

Kinetic Studies of the Effect of pH on the Trypsin-Catalyzed Hydrolysis of N-α-benzyloxycarbonyl-l-lysine-p-nitroanilide: Mechanism of Trypsin Catalysis.

The pH dependence of the trypsin-catalyzed hydrolysis of N-α-benzyloxycarbonyl-l-lysine p-nitroanilide has been studied at 25 °C. kcat/KM was maximal at alkaline pH values but decreased with decreasing pH. kcat/KM was dependent on free enzyme pKa values of 6.75 ± 0.09 and 4.10 ± 0.13, which were assigned to the ionization of the active site histidine-57 and aspartate-189, respectively. Protonation of either group abolished catalytic activity. kcat is shown to equal the acylation rate constant k2 over the pH range studied. k2 decreased on the protonation of two groups with pKa values of 4.81 ± 0.15 and 4.23 ± 0.19. We assign the pKa of 4.23 to the ionization of the aspartate-189 residue and the pKa of 4.81 to the oxyanion of the tetrahedral intermediate formed during acylation. We conclude that during acylation, breakdown of the catalytic tetrahedral intermediate is rate-limiting and that there is a strong interaction between the imidazolium ion of histidine-57 and the oxyanion of the catalytic tetrahedral intermediate, which perturbs their pKa values. From the pH dependence of k3, we conclude that deacylation depends on a pKa of 6.41 ± 0.22 and that the ionization of the carboxylate group of aspartate-189 does not have a significant effect on the rate of deacylation (k3). A catalytic mechanism is proposed to explain the pH dependence of catalysis.

PBP Isolation and DD-Carboxypeptidase Assay.

Penicillin-binding proteins (PBPs) share the namesake because of their ability to bind penicillin or any beta-lactam antibiotic. In other words, PBPs are the targets of β-lactam antibiotics that hold nearly 60% of the global antibiotic market. These enzymes catalyze the final stages of peptidoglycan (PG) biosynthesis by acting as transglycosylases and transpeptidases. PBPs are also involved in PG remodeling by catalyzing DD-carboxypeptidase (DD-CPase) and endopeptidase reactions. Though the cross-linking abilities of PBPs are well known, the process of remodeling is still unclear, thereby drawing attention toward the DD-CPase enzymes. Here, we describe the step-by-step procedures for isolation of the bacterial cell membrane and detection of PBPs in it, followed by the purification of PBPs (DD-CPases) by both ampicillin-affinity and nickel-nitrilotriacetic acid (Ni-NTA) chromatography. The protocols to determine the enzymatic efficiency are also elucidated. The assays are aimed to determine the kinetic parameters for the interaction of the PBP with BOCILLIN, to evaluate its acylation and deacylation rates, and with its peptide substrates, to assess its DD-CPase activity.