DOCSLIB.ORG

Avibactam Is a Covalent, Reversible, Non–Β-Lactam Β-Lactamase Inhibitor

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Avibactam Is a Covalent, Reversible, Non–Β-Lactam Β-Lactamase Inhibitor Avibactam is a covalent, reversible, non–β-lactam β-lactamase inhibitor David E. Ehmann1, Haris Jahic´, Philip L. Ross, Rong-Fang Gu, Jun Hu, Gunther Kern, Grant K. Walkup, and Stewart L. Fisher Infection Innovative Medicines Unit, AstraZeneca Research & Development Boston, Waltham, MA 02451 Edited by Vern L. Schramm, Albert Einstein College of Medicine, Bronx, NY, and approved June 5, 2012 (received for review March 26, 2012) Avibactam is a β-lactamase inhibitor that is in clinical develop- Gram-negative organisms (http://clinicaltrials.gov). A design ment, combined with β-lactam partners, for the treatment of bac- strategy for β-lactamase inhibitors has been to focus on scaffolds terial infections comprising Gram-negative organisms. Avibactam that maintain the capacity to rapidly acylate a wide range of is a structural class of inhibitor that does not contain a β-lactam β-lactamases while minimizing the liability of hydrolysis. Avi- core but maintains the capacity to covalently acylate its β-lacta- bactam has been described as possessing these desirable prop- mase targets. Using the TEM-1 enzyme, we characterized avibac- erties (9), but the molecular understanding of how it inhibits its tam inhibition by measuring the on-rate for acylation and the off- targets is not well-understood. rate for deacylation. The deacylation off-rate was 0.045 min−1, In this work, the detailed enzymatic mechanism of inhibition for which allowed investigation of the deacylation route from TEM-1. avibactam against the β-lactamase TEM-1 is described. Investi- Using NMR and MS, we showed that deacylation proceeds through gation of onset of and recovery from TEM-1 inhibition, coupled regeneration of intact avibactam and not hydrolysis. Other than with biophysical measurements of the enzyme–inhibitor complex, TEM-1, four additional clinically relevant β-lactamases were shown leads us to propose a model for covalent β-lactamase inhibition to release intact avibactam after being acylated. We showed that that is reversible but not susceptible to hydrolysis. Profiling of avibactam is a covalent, slowly reversible inhibitor, which is a unique additional clinically important class A and C β-lactamases sug- mechanism of inhibition among β-lactamase inhibitors. gests that this highly unusual reversible acylation and deacylation mechanism is a general mechanism of inhibition for avibactam. BIOCHEMISTRY antibacterial | drug discovery | enzymology Results β here is an urgent need for new antibacterial agents that are Onset of Acylation. The onset of inhibition of TEM-1 -lactamase active against drug-resistant bacteria. In particular, some by avibactam was investigated using conventional and stopped- T fl fl Gram-negative pathogens have accumulated enough resistance ow spectroscopy. Without stopped- ow spectroscopy, the ad- ∼ mechanisms to render them virtually untreatable by modern dition of enzyme resulted in a time lag of 3 s in our apparatus antibacterial chemotherapy (1, 2). A mainstay for treatment of and allowed investigation of avibactam concentrations up to μ fi Gram-negative infections is the β-lactam classes of drugs. The 2.5 M. Under these conditions, the observed pseudo rst-order most common form of resistance to β-lactam antibiotics is the rate constant for formation of inhibited enzyme, kobs, was not expression of various β-lactamase enzymes capable of hydrolyz- saturable with respect to the concentration of avibactam. Stop- fl ing the β-lactam ring of β-lactam drugs, rendering them in- ped- ow equipment allowed interrogation of shorter time regi- effective. As new β-lactams have been introduced into clinical mens from 250 ms to 7 s and higher avibactam concentrations μ use, a changing landscape of β-lactamases has been selected and from 8 to 50 M. The kobs values from both studies overlaid with disseminated. Presently, over 1,000 β-lactamases have been each other and remained linear at the highest avibactam con- documented comprising several structural classes and a wide centration (Fig. 2). Although this linear relationship is required range of substrate promiscuities and catalytic efficiencies (3, 4). of a one-step inhibition mechanism, it is also consistent with a In efforts to restore the efficacy of β-lactam antibiotics, β-lac- two-step binding and acylation process, where the initial binding β tamases have also been targeted with a variety of inhibitors (5, 6). constant is very weak (10). Covalent -lactamase inhibitors fol- The three inhibitors approved for clinical use are clavulanic acid, low a two-step binding and acylation reaction, and based on this tazobactam, and sulbactam, all of which contain a β-lactam core. A precedent, avibactam inhibition was modeled to a two-step challenge for the development of broad-spectrum β-lactamase in- mechanism as described in Materials and Methods. Fitting the plot of kobs vs. inhibitor concentration to an equation discussed hibitors is the mechanistic diversity in β-lactamase enzymes, with − − in Materials and Methods yielded a slope of 1.6 × 105 M 1s 1 the largest distinction being between the enzyme classes that use a 5 −1 −1 β (±0.1 × 10 M s at 95% confidence interval). For a two-step serine residue as the nucleophilic species and the metallo- -lac- fi tamases, which directly activate water for hydrolysis (7). A shared mechanism with a weak af nity for the initial encounter complex, β – the slope value determined in this manner is the second-order mechanistic feature of the marketed -lactam based inhibitors β is their reaction with the serine enzymes to form a covalent acyl- rate constant for -lactamase acylation. The contributing terms enzyme intermediate. On ring opening, the acyl-enzyme inter- for the noncovalent Ki and the ring-opening chemical step k2 mediate can undergo additional rearrangements or be released cannot be determined precisely (11). through hydrolysis to regenerate the active β-lactamase enzyme (8). Originally designed to combat class A serine β-lactamase enzymes such as TEM-1, the clinical use of β-lactam–based inhibitors has Author contributions: D.E.E., H.J., P.L.R., R.-F.G., J.H., G.K., G.K.W., and S.L.F. designed research; H.J., P.L.R., R.-F.G., J.H., G.K., and G.K.W. performed research; H.J., P.L.R., R.-F.G., been diminished by the emergence of enzymes against which they J.H., G.K., and G.K.W. analyzed data; and D.E.E. wrote the paper. are ineffective. Despite intense investigation by pharmaceutical Conflict of interest statement: All authors are present or past employees of AstraZeneca, companies, no new β-lactamase inhibitor has reached the market as stated in the affiliations, and potentially own stock and/or hold stock options in in over 19 years. the company. Avibactam (Fig. 1) is a member of a class of inhibitors called This article is a PNAS Direct Submission. the diazabicyclooctanes (DBOs) (9), and it is currently entering 1To whom correspondence should be addressed. E-mail: [email protected]. phases II and III clinical trials combined with ceftaroline and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ceftazidime for the treatment of serious infections caused by 1073/pnas.1205073109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1205073109 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 Fig. 1. Structures of β-lactamase inhibitors used in this study. Off-Rate of Deacylation. The experiments investigating the onset of observed. By following the return of activity in a continuous avibactam inhibition revealed that the off-rate for return of en- manner for 1 h at a much lower EI* concentration (25 pM as in zyme activity was much slower than the time regimen for onset of Fig. 2), the deacylation from TEM-1 can be measured. acylation. Therefore, a separate experiment was performed to measure the deacylation off-rate. The most common routes of Lack of Hydrolysis or Rearrangement. The recovery of enzyme ac- deacylation of β-lactamase inhibitors are through hydrolysis, tivity on dilution of the acylated enzyme led to experiments chemical rearrangement, or a combination of these paths (8). Off- designed to identify the deacylation routes involved. Avibactam rates for deacylation from TEM-1 were measured using a jump has been assumed to covalently acylate its β-lactamase target dilution method (12) comparing avibactam with clavulanic acid based on acyl-enzyme adducts seen in electrospray ionization MS and tazobactam. Avibactam displayed a slow return of activity (16). To eliminate the possibility of a tight noncovalent interaction, − with an off-rate of 0.045 ± 0.022 min 1, which converts to a resi- the acylated TEM-1 was subjected to strong denaturing conditions followed by MS. The retention of the acyl-enzyme adduct (Fig. S2) dence time half-life (t1/2)of16± 8 min (Fig. 3). In contrast, TEM- 1 with clavulanic acid displayed a partial return of activity, which under these conditions supports the covalent nature of this linkage. > is attributed to rearrangement from the acylated enzyme form to The observation that enzyme inhibition was maintained for 24 h μ additional irreversible acyl-enzyme species (13). TEM-1 in- at 1 M acyl-enzyme (Fig. S1) suggested that the carbamoyl acyl- hibition by tazobactam follows a branched deacylation pathway enzyme intermediate is stable to hydrolysis. This suggestion was fi 1 that favors hydrolysis over rearrangement (14), which in the off- con rmed by H NMR spectroscopy. As opposed to tazobactam, rate assay, manifested as a rapid return to nearly full activity. which was hydrolyzed by TEM-1 (Fig. 4A), avibactam remained The measured off-rate for avibactam suggested that slow intact after 24 h of incubation with a high concentration of TEM-1 deacylation through hydrolysis or reversibility was occurring, and (Fig.
Load more

Recommended publications
  • Electronic Supplementary Material (ESI) for Analyst. This Journal Is © the Royal Society of Chemistry 2017
    Electronic Supplementary Material (ESI) for Analyst. This journal is © The Royal Society of Chemistry 2017 Supplemental Table 2. Proteins Increased in Either Blood or Horizon media Table 2A. Proteins Increased in Spores Produced on Horizon Soil Over Spores Produced on Blood Medium quasi.fdr Protein Protein Class/Name KEGG Pathway Names or Function (if ID Pathways found in KEGG) Amino Acid Metabolism bat00250, bat00280, Alanine, aspartate and glutamate metabolism, bat00410, Valine, leucine and isoleucine degradation, bat00640, beta-Alanine metabolism to acetyl CoA, 4.50E-07 BAS0310 4-aminobutyrate aminotransferase bat00650 Propanoate metabolism, Butanoate metabolism bat00270, bat00330, Cysteine and methionine metabolism, Arginine bat00410, and proline metabolism, beta-Alanine 3.84E-10 BAS5060 spermidine synthase bat00480 metabolism, Glutathione metabolism bat00270, bat00330, Cysteine and methionine metabolism, Arginine bat00410, and proline metabolism, beta-Alanine 2.30E-09 BAS5219 spermidine synthase bat00480 metabolism, Glutathione metabolism bat00250, Alanine, aspartate and glutamate metabolism, 6.94E-07 BAS0561 alanine dehydrogenase bat00430 Taurine and hypotaurine metabolism bat00250, Alanine, aspartate and glutamate metabolism, 5.23E-07 BAS4521 alanine dehydrogenase bat00430 Taurine and hypotaurine metabolism 0.005627 BAS5218 agmatinase, putative bat00330 Arginine and proline metabolism 2,3,4,5-tetrahydropyridine-2- carboxylate N-succinyltransferase, 1.40E-10 BAS3891 putative bat00300 Lysine biosynthesis bat00010, bat00020, Glycolysis
    [Show full text]
  • Supplementary Table S1. Table 1. List of Bacterial Strains Used in This Study Suppl
    Supplementary Material Supplementary Tables: Supplementary Table S1. Table 1. List of bacterial strains used in this study Supplementary Table S2. List of plasmids used in this study Supplementary Table 3. List of primers used for mutagenesis of P. intermedia Supplementary Table 4. List of primers used for qRT-PCR analysis in P. intermedia Supplementary Table 5. List of the most highly upregulated genes in P. intermedia OxyR mutant Supplementary Table 6. List of the most highly downregulated genes in P. intermedia OxyR mutant Supplementary Table 7. List of the most highly upregulated genes in P. intermedia grown in iron-deplete conditions Supplementary Table 8. List of the most highly downregulated genes in P. intermedia grown in iron-deplete conditions Supplementary Figures: Supplementary Figure 1. Comparison of the genomic loci encoding OxyR in Prevotella species. Supplementary Figure 2. Distribution of SOD and glutathione peroxidase genes within the genus Prevotella. Supplementary Table S1. Bacterial strains Strain Description Source or reference P. intermedia V3147 Wild type OMA14 isolated from the (1) periodontal pocket of a Japanese patient with periodontitis V3203 OMA14 PIOMA14_I_0073(oxyR)::ermF This study E. coli XL-1 Blue Host strain for cloning Stratagene S17-1 RP-4-2-Tc::Mu aph::Tn7 recA, Smr (2) 1 Supplementary Table S2. Plasmids Plasmid Relevant property Source or reference pUC118 Takara pBSSK pNDR-Dual Clonetech pTCB Apr Tcr, E. coli-Bacteroides shuttle vector (3) plasmid pKD954 Contains the Porpyromonas gulae catalase (4)
    [Show full text]
  • Supporting Information
    Supporting Information Lee et al. 10.1073/pnas.0903619106 SI Materials and Methods The tobramycin sensitivities for strains expressing the rmtD Plasmids. Plasmid p(amgRSϩ) was constructed by inserting a 2.4 ribosomal methylase gene were determined by a 2-fold broth kb PCR fragment carrying the region from 206 bp upstream of dilution test. Inocula were prepared by growing cells at 37 °C for the amgR start codon to 60 bp downstream of the amgS 16 h in LB lacking NaCl. Cells were diluted to approximately ϫ 6 termination codon into a derivative of pUCP19 (1). Plasmid 3.3 10 cells/ml based on OD600. After a 90 min incubation at 37 °C, 10 ␮l aliquots were distributed to wells (in 96-well format) pRmtD was constructed by inserting the 2.3 kb HindIII-XbaI ␮ fragment carrying rmtD from pPA95B1 (2) into a derivative of containing 125 l LB buffered with 0.1 M MOPS at pH 7.6 pUCP19 (1). Plasmids used for complementation studies (Table containing different concentrations of tobramycin. MICs were 1) were constructed in pUCP19 derivatives by inserting PCR determined after incubation for 24 h at 37 °C. fragments corresponding to the genes tested. Plasmid pIbsC was Transcriptional Profiling. MPAO1 and ⌬amgRS transcriptomes constructed by inserting a 1.8 kb PCR fragment carrying the were compared under tobramycin-treated and -untreated con- arabinose-inducible ibsC construct (P -ibsC) from pAZ3-ibsC bad ditions. Samples were prepared in duplicate from independent (3) into a derivative of pUCP19 (4), resulting in pIbsC. The cultures grown in 15 ml LB lacking NaCl.
    [Show full text]
  • Generate Metabolic Map Poster
    Authors: Pallavi Subhraveti Anamika Kothari Quang Ong Ron Caspi An online version of this diagram is available at BioCyc.org. Biosynthetic pathways are positioned in the left of the cytoplasm, degradative pathways on the right, and reactions not assigned to any pathway are in the far right of the cytoplasm. Transporters and membrane proteins are shown on the membrane. Ingrid Keseler Peter D Karp Periplasmic (where appropriate) and extracellular reactions and proteins may also be shown. Pathways are colored according to their cellular function. Csac1394711Cyc: Candidatus Saccharibacteria bacterium RAAC3_TM7_1 Cellular Overview Connections between pathways are omitted for legibility. Tim Holland TM7C00001G0420 TM7C00001G0109 TM7C00001G0953 TM7C00001G0666 TM7C00001G0203 TM7C00001G0886 TM7C00001G0113 TM7C00001G0247 TM7C00001G0735 TM7C00001G0001 TM7C00001G0509 TM7C00001G0264 TM7C00001G0176 TM7C00001G0342 TM7C00001G0055 TM7C00001G0120 TM7C00001G0642 TM7C00001G0837 TM7C00001G0101 TM7C00001G0559 TM7C00001G0810 TM7C00001G0656 TM7C00001G0180 TM7C00001G0742 TM7C00001G0128 TM7C00001G0831 TM7C00001G0517 TM7C00001G0238 TM7C00001G0079 TM7C00001G0111 TM7C00001G0961 TM7C00001G0743 TM7C00001G0893 TM7C00001G0630 TM7C00001G0360 TM7C00001G0616 TM7C00001G0162 TM7C00001G0006 TM7C00001G0365 TM7C00001G0596 TM7C00001G0141 TM7C00001G0689 TM7C00001G0273 TM7C00001G0126 TM7C00001G0717 TM7C00001G0110 TM7C00001G0278 TM7C00001G0734 TM7C00001G0444 TM7C00001G0019 TM7C00001G0381 TM7C00001G0874 TM7C00001G0318 TM7C00001G0451 TM7C00001G0306 TM7C00001G0928 TM7C00001G0622 TM7C00001G0150 TM7C00001G0439 TM7C00001G0233 TM7C00001G0462 TM7C00001G0421 TM7C00001G0220 TM7C00001G0276 TM7C00001G0054 TM7C00001G0419 TM7C00001G0252 TM7C00001G0592 TM7C00001G0628 TM7C00001G0200 TM7C00001G0709 TM7C00001G0025 TM7C00001G0846 TM7C00001G0163 TM7C00001G0142 TM7C00001G0895 TM7C00001G0930 Detoxification Carbohydrate Biosynthesis DNA combined with a 2'- di-trans,octa-cis a 2'- Amino Acid Degradation an L-methionyl- TM7C00001G0190 superpathway of pyrimidine deoxyribonucleotides de novo biosynthesis (E.
    [Show full text]
  • Supplementary Table S4. FGA Co-Expressed Gene List in LUAD
    Supplementary Table S4. FGA co-expressed gene list in LUAD tumors Symbol R Locus Description FGG 0.919 4q28 fibrinogen gamma chain FGL1 0.635 8p22 fibrinogen-like 1 SLC7A2 0.536 8p22 solute carrier family 7 (cationic amino acid transporter, y+ system), member 2 DUSP4 0.521 8p12-p11 dual specificity phosphatase 4 HAL 0.51 12q22-q24.1histidine ammonia-lyase PDE4D 0.499 5q12 phosphodiesterase 4D, cAMP-specific FURIN 0.497 15q26.1 furin (paired basic amino acid cleaving enzyme) CPS1 0.49 2q35 carbamoyl-phosphate synthase 1, mitochondrial TESC 0.478 12q24.22 tescalcin INHA 0.465 2q35 inhibin, alpha S100P 0.461 4p16 S100 calcium binding protein P VPS37A 0.447 8p22 vacuolar protein sorting 37 homolog A (S. cerevisiae) SLC16A14 0.447 2q36.3 solute carrier family 16, member 14 PPARGC1A 0.443 4p15.1 peroxisome proliferator-activated receptor gamma, coactivator 1 alpha SIK1 0.435 21q22.3 salt-inducible kinase 1 IRS2 0.434 13q34 insulin receptor substrate 2 RND1 0.433 12q12 Rho family GTPase 1 HGD 0.433 3q13.33 homogentisate 1,2-dioxygenase PTP4A1 0.432 6q12 protein tyrosine phosphatase type IVA, member 1 C8orf4 0.428 8p11.2 chromosome 8 open reading frame 4 DDC 0.427 7p12.2 dopa decarboxylase (aromatic L-amino acid decarboxylase) TACC2 0.427 10q26 transforming, acidic coiled-coil containing protein 2 MUC13 0.422 3q21.2 mucin 13, cell surface associated C5 0.412 9q33-q34 complement component 5 NR4A2 0.412 2q22-q23 nuclear receptor subfamily 4, group A, member 2 EYS 0.411 6q12 eyes shut homolog (Drosophila) GPX2 0.406 14q24.1 glutathione peroxidase
    [Show full text]
  • Metligdb: a Web-Based Database for the Identification of Chemical Groups
    computer programs Journal of Applied MetLigDB: a web-based database for the Crystallography identification of chemical groups to design ISSN 0021-8898 metalloprotein inhibitors Received 12 April 2011 Accepted 10 June 2011 Hwanho Choi,‡ Hongsuk Kang‡ and Hwangseo Park* Department of Bioscience and Biotechnology, Sejong University, 98 Kunja-dong, Kwangjin-ku, Seoul 143-747, Korea. Correspondence e-mail: [email protected] MetLigDB (http://silver.sejong.ac.kr/MetLigDB) is a publicly accessible web- based database through which the interactions between a variety of chelating groups and various central metal ions in the active site of metalloproteins can be explored in detail. Additional information can also be retrieved, including protein and inhibitor names, the amino acid residues coordinated to the central metal ion, and the binding affinity of the inhibitor for the target metalloprotein. Although many metalloproteins have been considered promising targets for drug discovery, it is difficult to discover new inhibitors because of the difficulty in designing a suitable chelating moiety to impair the catalytic activity of the central metal ion. Because both common and specific chelating groups can be identified for varying metal ions and the associated coordination environments, # 2011 International Union of Crystallography MetLigDB is expected to give users insight into designing new inhibitors of Printed in Singapore – all rights reserved metalloproteins for drug discovery. 1. Introduction 2008), the coordination geometry around the metal ion (Castagnetto The importance of metal ions in biological systems is increasingly et al., 2002; Andreini, Bertini, Cavallaro, Holliday & Thornton, 2009), drawing attention, as indicated by the recent emergence of terms such catalytic mechanisms (Andreini, Bertini, Cavallaro, Najmanovich & as metallome and metallomics (Shi & Chance, 2008).
    [Show full text]
  • High Coverage Metabolomics Analysis Reveals Phage-Specific Alterations to Pseudomonas Aeruginosa Physiology During Infection
    The ISME Journal (2016) 10, 1823–1835 © 2016 International Society for Microbial Ecology All rights reserved 1751-7362/16 www.nature.com/ismej ORIGINAL ARTICLE High coverage metabolomics analysis reveals phage-specific alterations to Pseudomonas aeruginosa physiology during infection Jeroen De Smet1, Michael Zimmermann2, Maria Kogadeeva2, Pieter-Jan Ceyssens1,3, Wesley Vermaelen1, Bob Blasdel1, Ho Bin Jang1, Uwe Sauer2 and Rob Lavigne1 1Laboratory of Gene Technology, Department of Biosystems, KU Leuven, Heverlee, Belgium; 2Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule (ETH) Zürich, Zürich, Switzerland and 3Unit Bacterial Diseases, Scientific Institute of Public Health (WIV-ISP), Brussels, Belgium Phage-mediated metabolic changes in bacteria are hypothesized to markedly alter global nutrient and biogeochemical cycles. Despite their theoretic importance, experimental data on the net metabolic impact of phage infection on the bacterial metabolism remains scarce. In this study, we tracked the dynamics of intracellular metabolites using untargeted high coverage metabolomics in Pseudomo- nas aeruginosa cells infected with lytic bacteriophages from six distinct phage genera. Analysis of the metabolomics data indicates an active interference in the host metabolism. In general, phages elicit an increase in pyrimidine and nucleotide sugar metabolism. Furthermore, clear phage-specific and infection stage-specific responses are observed, ranging from extreme metabolite depletion (for example, phage YuA) to complete reorganization of the metabolism (for example, phage phiKZ). As expected, pathways targeted by the phage-encoded auxiliary metabolic genes (AMGs) were enriched among the metabolites changing during infection. The effect on pyrimidine metabolism of phages encoding AMGs capable of host genome degradation (for example, YuA and LUZ19) was distinct from those lacking nuclease-encoding genes (for example, phiKZ), which demonstrates the link between the encoded set of AMGs of a phage and its impact on host physiology.
    [Show full text]
  • The Microbiota-Produced N-Formyl Peptide Fmlf Promotes Obesity-Induced Glucose
    Page 1 of 230 Diabetes Title: The microbiota-produced N-formyl peptide fMLF promotes obesity-induced glucose intolerance Joshua Wollam1, Matthew Riopel1, Yong-Jiang Xu1,2, Andrew M. F. Johnson1, Jachelle M. Ofrecio1, Wei Ying1, Dalila El Ouarrat1, Luisa S. Chan3, Andrew W. Han3, Nadir A. Mahmood3, Caitlin N. Ryan3, Yun Sok Lee1, Jeramie D. Watrous1,2, Mahendra D. Chordia4, Dongfeng Pan4, Mohit Jain1,2, Jerrold M. Olefsky1 * Affiliations: 1 Division of Endocrinology & Metabolism, Department of Medicine, University of California, San Diego, La Jolla, California, USA. 2 Department of Pharmacology, University of California, San Diego, La Jolla, California, USA. 3 Second Genome, Inc., South San Francisco, California, USA. 4 Department of Radiology and Medical Imaging, University of Virginia, Charlottesville, VA, USA. * Correspondence to: 858-534-2230, [email protected] Word Count: 4749 Figures: 6 Supplemental Figures: 11 Supplemental Tables: 5 1 Diabetes Publish Ahead of Print, published online April 22, 2019 Diabetes Page 2 of 230 ABSTRACT The composition of the gastrointestinal (GI) microbiota and associated metabolites changes dramatically with diet and the development of obesity. Although many correlations have been described, specific mechanistic links between these changes and glucose homeostasis remain to be defined. Here we show that blood and intestinal levels of the microbiota-produced N-formyl peptide, formyl-methionyl-leucyl-phenylalanine (fMLF), are elevated in high fat diet (HFD)- induced obese mice. Genetic or pharmacological inhibition of the N-formyl peptide receptor Fpr1 leads to increased insulin levels and improved glucose tolerance, dependent upon glucagon- like peptide-1 (GLP-1). Obese Fpr1-knockout (Fpr1-KO) mice also display an altered microbiome, exemplifying the dynamic relationship between host metabolism and microbiota.
    [Show full text]
  • Discovery of a New Genetic Variant of Methionine Aminopeptidase From
    Discovery of a New Genetic Variant of Methionine Aminopeptidase from Streptococci with Possible Post- Translational Modifications: Biochemical and Structural Characterization Tarun Arya, Chandan Kishor, Venkateshwarlu Saddanapu, Ravikumar Reddi, Anthony Addlagatta* Centre for Chemical Biology, CSIR-Indian Institute of Chemical Technology, Hyderabad, Andhra Pradesh, India Abstract Protein N-terminal methionine excision is an essential co-translational process that occurs in the cytoplasm of all organisms. About 60-70% of the newly synthesized proteins undergo this modification. Enzyme responsible for the removal of initiator methionine is methionine aminopeptidase (MetAP), which is a dinuclear metalloprotease. This protein is conserved through all forms of life from bacteria to human except viruses. MetAP is classified into two isoforms, Type I and II. Removal of the map gene or chemical inhibition is lethal to bacteria and to human cell lines, suggesting that MetAP could be a good drug target. In the present study we describe the discovery of a new genetic variant of the Type I MetAP that is present predominantly in the streptococci bacteria. There are two inserts (insert one: 27 amino acids and insert two: four residues) within the catalytic domain. Possible glycosylation and phosphorylation posttranslational modification sites are identified in the ‘insert one’. Biochemical characterization suggests that this enzyme behaves similar to other MetAPs in terms of substrate specificity. Crystal structure Type Ia MetAP from Streptococcus pneumoniae (SpMetAP1a) revealed that it contains two molecules in the asymmetric unit and well ordered inserts with structural features that corroborate the possible posttranslational modification. Both the new inserts found in the SpMetAP1a structurally align with the P-X-X-P motif found in the M.
    [Show full text]
  • Metalloenzymes: Native Co-Factor Or Experimental Artifact? Marcy Hernick* Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA
    nalytic A al & B y i tr o s c i h e Hernick, Biochem Anal Biochem 2012, 1:6 m m e Biochemistry & i h s c t r o DOI: 10.4172/2161-1009.1000e120 i y B ISSN: 2161-1009 Analytical Biochemistry Editorial Open Access Metalloenzymes: Native Co-factor or Experimental Artifact? Marcy Hernick* Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA There has been much interest in the biochemical characterization metallohydrolases. Superoxide dismutase (SOD) is an example of a of metalloenzymes, owing in part to past successes in targeting metalloenzyme that utilizes Mn2+, Fe2+, or Mn/Fe as native co-factors metalloenzymes for the development of therapeutic agents. It is across different species [16-20]. (Note: The cited references offer just a estimated that ~10% of approved drugs target metalloenzymes, [1] such small sampling of the research examining SOD co-factor preferences). as carbonic anhydrase, matrix metalloprotesases, and angiotensin- These detailed studies on SOD reinforce the important contributions converting enzyme [2-5]. Inhibitors of metalloenzymes typically that metal ion availability and experimental conditions have on contain a metal-binding group that targets the catalytic metal ion, identifying the native co-factor(s) preferences of metalloenzymes. and therefore, inhibitor affinity is affected by changes to the catalytic In addition to metalloenzymes that prefer a single co-factor as metal ion. Consequently, the development of therapeutically effective described above, there are also increasing numbers of cambialistic inhibitors requires that the biologically relevant form(s) of the enzyme enzymes that can utilize multiple co-factors in vivo has been identified.
    [Show full text]
  • Exploration of the Core Metabolism of Symbiotic Bacteria
    Klein et al. BMC Genomics 2012, 13:438 http://www.biomedcentral.com/1471-2164/13/438 RESEARCH ARTICLE Open Access Exploration of the core metabolism of symbiotic bacteria Cecilia Coimbra Klein1,2,5*, Ludovic Cottret3, Janice Kielbassa1,2, Hubert Charles1,4, Christian Gautier1,2, Ana Tereza Ribeiro de Vasconcelos1,2,5, Vincent Lacroix1,2 and Marie-France Sagot1,2* Abstract Background: A large number of genome-scale metabolic networks is now available for many organisms, mostly bacteria. Previous works on minimal gene sets, when analysing host-dependent bacteria, found small common sets of metabolic genes. When such analyses are restricted to bacteria with similar lifestyles, larger portions of metabolism are expected to be shared and their composition is worth investigating. Here we report a comparative analysis of the small molecule metabolism of symbiotic bacteria, exploring common and variable portions as well as the contribution of different lifestyle groups to the reduction of a common set of metabolic capabilities. Results: We found no reaction shared by all the bacteria analysed. Disregarding those with the smallest genomes, we still do not find a reaction core, however we did find a core of biochemical capabilities. While obligate intracellular symbionts have no core of reactions within their group, extracellular and cell-associated symbionts do have a small core composed of disconnected fragments. In agreement with previous findings in Escherichia coli, their cores are enriched in biosynthetic processes whereas the variable metabolisms have similar ratios of biosynthetic and degradation reactions. Conversely, the variable metabolism of obligate intracellular symbionts is enriched in anabolism. Conclusion: Even when removing the symbionts with the most reduced genomes, there is no core of reactions common to the analysed symbiotic bacteria.
    [Show full text]
  • Surface Analyses and Immune Reactivities of Major Cell Wall-Associated Proteins of Group a Streptococcus Jason N
    INFECTION AND IMMUNITY, May 2005, p. 3137–3146 Vol. 73, No. 5 0019-9567/05/$08.00ϩ0 doi:10.1128/IAI.73.5.3137–3146.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved. Surface Analyses and Immune Reactivities of Major Cell Wall-Associated Proteins of Group A Streptococcus Jason N. Cole,1 Ruben D. Ramirez,1 Bart J. Currie,2 Stuart J. Cordwell,3 Steven P. Djordjevic,4 and Mark J. Walker1* School of Biological Sciences, University of Wollongong, Wollongong, NSW,1 Menzies School Downloaded from of Health Research, Charles Darwin University, Darwin, NT,2 Australian Proteome Analysis Facility, Macquarie University, Sydney, NSW,3 and NSW Agriculture, Elizabeth Macarthur Agricultural Institute, Camden, NSW,4 Australia Received 9 December 2004/Accepted 27 December 2004 A proteomic analysis was undertaken to identify cell wall-associated proteins of Streptococcus pyogenes. Seventy-four distinct cell wall-associated proteins were identified, 66 of which were novel. Thirty-three proteins were immunoreactive with pooled S. pyogenes-reactive human antisera. Biotinylation of the GAS cell surface http://iai.asm.org/ identified 23 cell wall-associated proteins that are surface exposed. The gram-positive human pathogen Streptococcus pyogenes undergone posttranslational modifications. It remains to be (group A streptococcus; GAS) is the etiologic agent of numer- determined whether or not these modifications are physiolog- ous suppurative diseases, ranging from mild skin infections, ical or an artifact caused by urea carbamylation, deamidation, such as pharyngitis, scarlet fever, impetigo, and cellulitis, to or immobilized pH gradient strip overload. With the exception severe invasive diseases such as septicemia, streptococcal toxic of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) on October 19, 2015 by University of Queensland Library shock syndrome, and necrotizing fasciitis (8).
    [Show full text]