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.

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Molecular Determinants of Substrate Recognition in Hematopoietic Protein-tyrosine Phosphatase

Journal of Biological Chemistry ◽

10.1074/jbc.m407820200 ◽

2004 ◽

Vol 279 (50) ◽

pp. 52150-52159 ◽

Cited By ~ 19

Author(s):

Zhonghui Huang ◽

Bo Zhou ◽

Zhong-Yin Zhang

Keyword(s):

Substrate Specificity ◽

Protein Tyrosine Phosphatase ◽

Cellular Proliferation ◽

High Efficiency ◽

Tyrosine Phosphatase ◽

Substrate Recognition ◽

Fold Increase ◽

Phosphoryl Transfer ◽

Protein Tyrosine ◽

Hydrolysis Of

The extracellular signal-regulated protein kinase 2 (ERK2) plays a central role in cellular proliferation and differentiation. Full activation of ERK2 requires dual phosphorylation of Thr183and Tyr185in the activation loop. Tyr185dephosphorylation by the hematopoietic protein-tyrosine phosphatase (HePTP) represents an important mechanism for down-regulating ERK2 activity. The bisphosphorylated ERK2 is a highly efficient substrate for HePTP with akcat/Kmof 2.6 × 106m–1s–1. In contrast, thekcatK/mvalues for the HePTP-catalyzed hydrolysis of Tyr(P) peptides are 3 orders of magnitude lower. To gain insight into the molecular basis for HePTP substrate specificity, we analyzed the effects of altering structural features unique to HePTP on the HePTP-catalyzed hydrolysis ofp-nitrophenyl phosphate, Tyr(P) peptides, and its physiological substrate ERK2. Our results suggest that substrate specificity is conferred upon HePTP by both negative and positive selections. To avoid nonspecific tyrosine dephosphorylation, HePTP employs Thr106in the substrate recognition loop as a key negative determinant to restrain its protein-tyrosine phosphatase activity. The extremely high efficiency and fidelity of ERK2 dephosphorylation by HePTP is achieved by a bipartite protein-protein interaction mechanism, in which docking interactions between the kinase interaction motif in HePTP and the common docking site in ERK2 promote the HePTP-catalyzed ERK2 dephosphorylation (∼20-fold increase inkcat/Km) by increasing the local substrate concentration, and second site interactions between the HePTP catalytic site and the ERK2 substrate-binding region enhance catalysis (∼20-fold increase inkcat/Km) by organizing the catalytic residues with respect to Tyr(P)185for optimal phosphoryl transfer.

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Rapid Screen for Tyrosine Kinase Inhibitor Resistance Mutations and Substrate Specificity

ACS Chemical Biology ◽

10.1021/acschembio.9b00283 ◽

2019 ◽

Vol 14 (9) ◽

pp. 1888-1895 ◽

Cited By ~ 1

Author(s):

Joseph M. Taft ◽

Sandrine Georgeon ◽

Chris Allen ◽

Sina Reckel ◽

Joseph DeSautelle ◽

...

Keyword(s):

Substrate Specificity ◽

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitor ◽

Kinase Inhibitor ◽

Resistance Mutations ◽

Inhibitor Resistance

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Role of Cathepsin A and Lysosomes in the Intracellular Activation of Novel Antipapillomavirus Agent GS-9191

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01603-10 ◽

2011 ◽

Vol 55 (5) ◽

pp. 2166-2173 ◽

Cited By ~ 17

Author(s):

Gabriel Birkus ◽

Nilima Kutty ◽

Christian R. Frey ◽

Riri Shribata ◽

Tsuifen Chou ◽

...

Keyword(s):

Fold Increase ◽

Cell Extract ◽

Intracellular Accumulation ◽

Nucleotide Analog ◽

Siha Cells ◽

Interfering Rna ◽

Rate Limiting ◽

Hydrolysis Of

ABSTRACTGS-9191, a bis-amidate prodrug of the nucleotide analog 9-(2-phosphonylmethoxyethyl)-N6-cyclopropyl-2,6-diaminopurine (cPrPMEDAP), was designed as a topical agent for the treatment of papillomavirus-associated proliferative disorders, such as genital warts. In this study, we investigated the mechanism of conversion of GS-9191 to cPrPMEDAP. We observed that GS-9191 is hydrolyzed in the presence of the lysosomal carboxypeptidase cathepsin A (CatA)in vitroand is less efficiently metabolized in CatA-deficient fibroblasts than in control cells. In addition, knockdown of CatA by small interfering RNA (siRNA) reduced the intracellular accumulation of GS-9191 metabolites. However, intracellular CatA levels did not correlate with the susceptibility of tested cell lines to GS-9191, indicating that the CatA step is unlikely to be rate limiting for the activation of GS-9191. Further analysis showed that upon the hydrolysis of the carboxylester bond in one of the GS-9191 amidate moieties, the unmasked carboxyl group displacesl-phenylalanine 2-methylpropyl ester from the other amidate moiety. The cPrPMEDAP–l-phenylalanine conjugate (cPrPMEDAP-Phe) formed is not metabolized by Hint1 (histidine triad nucleotide binding protein 1) phosphoramidase but undergoes spontaneous degradation to cPrPMEDAP in acidic pH that can be significantly enhanced by the addition of SiHa cell extract. Pretreatment of SiHa cells with bafilomycin A or chloroquine resulted in an 8-fold increase in the intracellular concentration of cPrPMEDAP-Phe metabolite and the accumulation of GS-9191 metabolites in the lysosomal/endosomal fraction. Together, these observations indicate that the conversion of GS-9191 to cPrPMEDAP occurs in lysosomes via CatA-mediated ester cleavage, followed by the release of cPrPMEDAP, most likely through the combination of enzyme-driven and spontaneous pH-driven hydrolysis of a cPrPMEDAP-Phe intermediate.

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Is perioperative antibiotic necessary in straightforward implant placement procedures?

Journal of Oral Implantology ◽

10.1563/aaid-joi-d-19-00282 ◽

2020 ◽

Author(s):

Elçin Bedeloğlu ◽

Mustafa Yalçın ◽

Cenker Zeki Koyuncuoğlu

Keyword(s):

Clavulanic Acid ◽

Dental Implant ◽

Prophylactic Antibiotic ◽

Antibiotic Use ◽

Implant Failure ◽

Control Group ◽

Implant Placement ◽

Significant Difference ◽

Amoxicillin Clavulanic Acid ◽

Experimental Group

The purpose of this non-random retrospective cohort study was to evaluate the impact of prophylactic antibiotic on early outcomes including postoperative pain, swelling, bleeding and cyanosis in patients undergoing dental implant placement before prosthetic loading. Seventy-five patients (45 males, 30 females) whose dental implant placement were completed, included to the study. Patients used prophylactic antibiotics were defined as the experimental group and those who did not, were defined as the control group. The experimental group received 2 g amoxicillin + clavulanic acid 1 h preoperatively and 1 g amoxicillin + clavulanic acid twice a day for 5 days postoperatively while the control group had received no prophylactic antibiotic therapy perioperatively. Data on pain, swelling, bleeding, cyanosis, flap dehiscence, suppuration and implant failure were analyzed on postoperative days 2, 7, and 14 and week 12. No statistically significant difference was detected between the two groups with regard to pain and swelling on postoperative days 2, 7, and 14 and week 12 ( p >0.05), while the severity of pain and swelling were greater on day 2 compared to day 7 and 14 and week 12 in both groups ( p =0.001 and p <0.05, respectively). Similarly, no significant difference was found between the two groups with regard to postoperative bleeding and cyanosis. Although flap dehiscence was more severe on day 7 in the experimental group, no significant difference was found between the two groups with regard to the percentage of flap dehiscence assessed at other time points. Within limitations of the study, it has been demonstrated that antibiotic use has no effect on implant failure rates in dental implant surgery with a limited number of implants. We conclude that perioperative antibiotic use may not be required in straightforward implant placement procedures. Further randomized control clinical studies with higher numbers of patients and implants are needed to substantiate our findings.

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Global patterns in genomic diversity underpinning the evolution of insecticide resistance in the aphid crop pest Myzus persicae

Communications Biology ◽

10.1038/s42003-021-02373-x ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Kumar Saurabh Singh ◽

Erick M. G. Cordeiro ◽

Bartlomiej J. Troczka ◽

Adam Pym ◽

Joanna Mackisack ◽

...

Keyword(s):

Host Plant ◽

Myzus Persicae ◽

Resistance Mechanisms ◽

Genomic Diversity ◽

Resistance Mutations ◽

Host Plant Adaptation ◽

Repeated Evolution ◽

Evolution Of Resistance ◽

Selective Forces ◽

Clonal Lines

AbstractThe aphid Myzus persicae is a destructive agricultural pest that displays an exceptional ability to develop resistance to both natural and synthetic insecticides. To investigate the evolution of resistance in this species we generated a chromosome-scale genome assembly and living panel of >110 fully sequenced globally sampled clonal lines. Our analyses reveal a remarkable diversity of resistance mutations segregating in global populations of M. persicae. We show that the emergence and spread of these mechanisms is influenced by host–plant associations, uncovering the widespread co‐option of a host-plant adaptation that also offers resistance against synthetic insecticides. We identify both the repeated evolution of independent resistance mutations at the same locus, and multiple instances of the evolution of novel resistance mechanisms against key insecticides. Our findings provide fundamental insights into the genomic responses of global insect populations to strong selective forces, and hold practical relevance for the control of pests and parasites.

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No difference in HIV-1 integrase inhibitor resistance between CSF and blood compartments

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkab064 ◽

2021 ◽

Author(s):

Basma Abdi ◽

Mouna Chebbi ◽

Marc Wirden ◽

Elisa Teyssou ◽

Sophie Sayon ◽

...

Keyword(s):

Integrase Inhibitor ◽

Routine Care ◽

Biological Data ◽

Recombinant Form ◽

Hiv Integrase ◽

Resistance Mutations ◽

Clinical Routine ◽

Inhibitor Resistance ◽

Hiv 1

Abstract Background Little is known about HIV-1 integrase inhibitor resistance in the CNS. Objectives This study aimed to evaluate integrase inhibitor resistance in CSF, as a marker of the CNS, and compare it with the resistance in plasma. Methods HIV integrase was sequenced both in plasma and CSF for 59 HIV-1 patients. The clinical and biological data were collected from clinical routine care. Results Among the 59 HIV-1 patients, 32 (54.2%) were under antiretroviral (ARV) treatment. The median (IQR) HIV-1 RNA in the plasma of viraemic patients was 5.32 (3.85–5.80) and 3.59 (2.16–4.50) log10 copies/mL versus 4.79 (3.56–5.25) and 3.80 (2.68–4.33) log10 copies/mL in the CSF of ARV-naive and ARV-treated patients, respectively. The patients were mainly infected with non-B subtypes (72.2%) with the most prevalent recombinant form being CRF02_AG (42.4%). The HIV-1 integrase sequences from CSF presented resistance mutations for 9/27 (33.3%) and 8/32 (25.0%) for ARV-naive (L74I, n = 3; L74I/M, n = 1; T97A, n = 1; E157Q, n = 4) and ARV-treated (L74I, n = 6; L74M, n = 1; T97A, n = 1; N155H, n = 1) patients, respectively. Integrase inhibitor resistance mutations in CSF were similar to those in plasma, except for 1/59 patients. Conclusions This work shows similar integrase inhibitor resistance profiles in the CNS and plasma in a population of HIV-1 viraemic patients.

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Erratum to: “Self-assembling catalytic systems based on new amphiphile containing purine fragment, exhibiting substrate specificity in hydrolysis of phosphorus acids esters”

Russian Journal of General Chemistry ◽

10.1134/s1070363217070350 ◽

2017 ◽

Vol 87 (7) ◽

pp. 1649-1649

Author(s):

D. A. Samarkina ◽

D. R. Gabdrakhmanov ◽

V. E. Semenov ◽

F. G. Valeeva ◽

L. M. Gubaidullina ◽

...

Keyword(s):

Substrate Specificity ◽

Catalytic Systems ◽

Self Assembling ◽

Hydrolysis Of

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Post-Operative Infection Following Hand Surgery

Journal of Hand Surgery (European Volume) ◽

10.1016/s0266-7681(05)80137-8 ◽

1995 ◽

Vol 20 (5) ◽

pp. 685-690 ◽

Cited By ~ 41

Author(s):

A. J. PLATT ◽

R. E. PAGE

Keyword(s):

Wound Infection ◽

Infection Rate ◽

Hand Surgery ◽

Fold Increase ◽

Antibiotic Use ◽

Postal Questionnaire ◽

British Society ◽

Antibiotic Administration ◽

Clinical Criteria ◽

Operative Wound

An audit was designed to analyse the risk factors for developing post-operative wound infection following hand surgery. 249 consecutive patients were prospectively entered into the study. 236 (95%) patients were available for follow-up. Infection was diagnosed by clinical criteria. There was an infection rate of 10.7% in elective operations and 9.7% in emergency operations. There was no significant reduction in infection rate in the elective group with the use of antibiotics ( P=0.5). In the emergency group of patients peri-operative antibiotic administration was associated with an 8.5-fold reduction in infection rate ( P=0.014). The presence of a dirty wound was associated with a 13.4-fold increase in post-operative wound infection rate ( P=0.002). A postal questionnaire of members of the British Society for Surgery of the Hand revealed a wide variation in antibiotic usage. Guidelines for antibiotic use in patients undergoing hand surgery are presented.

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