The discovery of penta-peptides inhibiting the activity of the formylglycine-generating enzyme and their potential antibacterial effects against Mycobacterium tuberculosis.
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ABSTRACT:
SUBMITTER: Asiimwe N
PROVIDER: S-EPMC9241360 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Type I sulfatases require an unusual co- or post-translational modification for their activity in hydrolyzing sulfate esters. In eukaryotic sulfatases, an active site cysteine residue is oxidized to the aldehyde-containing C(alpha)-formylglycine residue by the formylglycine-generating enzyme (FGE). The machinery responsible for sulfatase activation is poorly understood in prokaryotes. Here we describe the identification of a prokaryotic FGE from Mycobacterium tuberculosis. In addition, we solved the crystal structure of the Streptomyces coelicolor FGE homolog to 2.1 A resolution. The prokaryotic homolog exhibits remarkable structural similarity to human FGE, including the position of catalytic cysteine residues. Both biochemical and structural data indicate the presence of an oxidized cysteine modification in the active site that may be relevant to catalysis. In addition, we generated a mutant M. tuberculosis strain lacking FGE. Although global sulfatase activity was reduced in the mutant, a significant amount of residual sulfatase activity suggests the presence of FGE-independent sulfatases in this organism.
| S-EPMC2459300 | biostudies-literature
Project description:Formylglycine-generating enzyme (FGE) post-translationally converts a specific cysteine in newly synthesized sulfatases to formylglycine (FGly). FGly is the key catalytic residue of the sulfatase family, comprising 17 nonredundant enzymes in human that play essential roles in development and homeostasis. FGE, a resident protein of the endoplasmic reticulum, is also secreted. A major fraction of secreted FGE is N-terminally truncated, lacking residues 34-72. Here we demonstrate that this truncated form is generated intracellularly by limited proteolysis mediated by proprotein convertase(s) (PCs) along the secretory pathway. The cleavage site is represented by the sequence RYSR(72)?, a motif that is conserved in higher eukaryotic FGEs, implying important functionality. Residues Arg-69 and Arg-72 are critical because their mutation abolishes FGE processing. Furthermore, residues Tyr-70 and Ser-71 confer an unusual property to the cleavage motif such that endogenous as well as overexpressed FGE is only partially processed. FGE is cleaved by furin, PACE4, and PC5a. Processing is disabled in furin-deficient cells but fully restored upon transient furin expression, indicating that furin is the major protease cleaving FGE. Processing by endogenous furin occurs mostly intracellularly, although also extracellular processing is observed in HEK293 cells. Interestingly, the truncated form of secreted FGE no longer possesses FGly-generating activity, whereas the unprocessed form of secreted FGE is active. As always both forms are secreted, we postulate that furin-mediated processing of FGE during secretion is a physiological means of higher eukaryotic cells to regulate FGE activity upon exit from the endoplasmic reticulum.
| S-EPMC3581403 | biostudies-literature
Project description:Formylglycine-generating enzyme (FGE) is an O2 -utilizing oxidase that converts specific cysteine residues of client proteins to formylglycine. We show that CuI is an integral cofactor of this enzyme and binds with high affinity (KD =of 10-17 m) to a pair of active-site cysteines. These findings establish FGE as a novel type of copper enzyme.
| S-EPMC5324649 | biostudies-literature
Project description:To further our aim of synthesizing aldehyde-tagged proteins for research and biotechnology applications, we developed methods for recombinant production of aerobic formylglycine-generating enzyme (FGE) in good yield. We then optimized the FGE biocatalytic reaction conditions for conversion of cysteine to formylglycine in aldehyde tags on intact monoclonal antibodies. During the development of these conditions, we discovered that pretreating FGE with copper(II) is required for high turnover rates and yields. After further investigation, we confirmed that both aerobic prokaryotic (Streptomyces coelicolor) and eukaryotic (Homo sapiens) FGEs contain a copper cofactor. The complete kinetic parameters for both forms of FGE are described, along with a proposed mechanism for FGE catalysis that accounts for the copper-dependent activity.
| S-EPMC4505483 | biostudies-literature
Project description:The formylglycine (FGly)-generating enzyme (FGE) uses molecular oxygen to oxidize a conserved cysteine residue in all eukaryotic sulfatases to the catalytically active FGly. Sulfatases degrade and remodel sulfate esters, and inactivity of FGE results in multiple sulfatase deficiency, a fatal disease. The previously determined FGE crystal structure revealed two crucial cysteine residues in the active site, one of which was thought to be implicated in substrate binding. The other cysteine residue partakes in a novel oxygenase mechanism that does not rely on any cofactors. Here, we present crystal structures of the individual FGE cysteine mutants and employ chemical probing of wild-type FGE, which defined the cysteines to differ strongly in their reactivity. This striking difference in reactivity is explained by the distinct roles of these cysteine residues in the catalytic mechanism. Hitherto, an enzyme-substrate complex as an essential cornerstone for the structural evaluation of the FGly formation mechanism has remained elusive. We also present two FGE-substrate complexes with pentamer and heptamer peptides that mimic sulfatases. The peptides isolate a small cavity that is a likely binding site for molecular oxygen and could host reactive oxygen intermediates during cysteine oxidation. Importantly, these FGE-peptide complexes directly unveil the molecular bases of FGE substrate binding and specificity. Because of the conserved nature of FGE sequences in other organisms, this binding mechanism is of general validity. Furthermore, several disease-causing mutations in both FGE and sulfatases are explained by this binding mechanism.
| S-EPMC1324989 | biostudies-literature
Project description:The lack of effective therapies for treating tuberculosis (TB) is a global health problem. While Mycobacterium tuberculosis is notoriously resistant to most available antibiotics, we identified synthetic short cationic antimicrobial peptides that were active at low micromolar concentrations (less than 10 μM). These small peptides (averaging 10 amino acids) had remarkably broad spectra of antimicrobial activities against both bacterial and fungal pathogens and an indication of low cytotoxicity. In addition, their antimicrobial activities displayed various degrees of species specificity that were not related to taxonomy. For example, Candida albicans and Staphylococcus aureus were the best surrogates to predict peptide activity against M. tuberculosis, while Mycobacterium smegmatis was a poor surrogate. Principle component analysis of activity spectrum profiles identified unique features associated with activity against M. tuberculosis that reflect their distinctive amino acid composition; active peptides were more hydrophobic and cationic, reflecting increased tryptophan with compensating decreases in valine and other uncharged amino acids and increased lysine. These studies provide foundations for development of cationic antimicrobial peptides as potential new therapeutic agents for TB treatment.
| S-EPMC3632949 | biostudies-literature
Project description:Mycobacterium tuberculosis, the etiological agent of the infectious disease tuberculosis, kills approximately 1.5?million people annually, while the spread of multidrug-resistant strains is of great global concern. Thus, continuous efforts to identify new antitubercular drugs as well as novel targets are crucial. Recently, two prodrugs activated by the monooxygenase EthA, 7947882 and 7904688, which target the CTP synthetase PyrG, were identified and characterized. In this work, microbiological, biochemical, and in silico methodologies were used to demonstrate that both prodrugs possess a second target, the pantothenate kinase PanK. This enzyme is involved in coenzyme A biosynthesis, an essential pathway for M. tuberculosis growth. Moreover, compound 11426026, the active metabolite of 7947882, was demonstrated to directly inhibit PanK, as well. In an independent screen of a compound library against PyrG, two additional inhibitors were also found to be active against PanK. In conclusion, these direct PyrG and PanK inhibitors can be considered as leads for multitarget antitubercular drugs and these two enzymes could be employed as a "double-tool" in order to find additional hit compounds.
| S-EPMC5816626 | biostudies-literature
Project description:Bacteria commonly adapt to stresses by altering gene expression. To understand the response of M. tuberculosis (MTB) to various antibacterial agents, we performed transcriptomics on MTB bacilli exposed to several test compounds as well as known drugs (capreomycin, cycloserine, ethionamide, isoniazid, kanamycin, moxifloxacin, PA-824, rifampicin, streptomycin). Bacteria were exposed for 16 hrs to various concentrations of each drug (different multiples of the compound's MIC), as noted in the title of each sample. RNA was isolated and applied to arrays provided by TIGR under the NIAID contract N01-AI-15447
2015-11-11 | E-GEOD-71200 | biostudies-arrayexpress
Project description:The formylglycine-generating enzyme (FGE) is required for the posttranslational activation of type I sulfatases by oxidation of an active-site cysteine to C?-formylglycine. FGE has emerged as an enabling biotechnology tool due to the robust utility of the aldehyde product as a bioconjugation handle in recombinant proteins. Here, we show that Cu(I)-FGE is functional in O2 activation and reveal a high-resolution X-ray crystal structure of FGE in complex with its catalytic copper cofactor. We establish that the copper atom is coordinated by two active-site cysteine residues in a nearly linear geometry, supporting and extending prior biochemical and structural data. The active cuprous FGE complex was interrogated directly by X-ray absorption spectroscopy. These data unambiguously establish the configuration of the resting enzyme metal center and, importantly, reveal the formation of a three-coordinate tris(thiolate) trigonal planar complex upon substrate binding as furthermore supported by density functional theory (DFT) calculations. Critically, inner-sphere substrate coordination turns on O2 activation at the copper center. These collective results provide a detailed mechanistic framework for understanding why nature chose this structurally unique monocopper active site to catalyze oxidase chemistry for sulfatase activation.
| S-EPMC6431200 | biostudies-literature
Project description:Bacteria commonly adapt to stresses by altering gene expression. To understand the response of M. tuberculosis (MTB) to various antibacterial agents, we performed transcriptomics on MTB bacilli exposed to several test compounds as well as known drugs (capreomycin, cycloserine, ethionamide, isoniazid, kanamycin, moxifloxacin, PA-824, rifampicin, streptomycin).
2015-11-11 | GSE71200 | GEO