Biochimica et Biophysica Acta (BBA) - protein structure and molecular enzymology
Volume 1477, Issues 1–2,
March 7, 2000
, pages 157-167
Biochimica et Biophy...
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Removal of the N-terminal methionine from proteins and peptides depends on a new class of proteases represented by the binuclear metalloenzyme methionine aminopeptidaseEscherichia coli(eMetAP). Significant progress has recently been made in determining the structures of several members of this family. The identification of human MetAP as a target of putative anticancer drugs underscores the importance of this family of enzymes. Determining the connection pathsE coliMetAP of a substrate-like bestatin-based inhibitor and phosphorus-containing transition-state analogues and reaction products rationalized the substrate specificity and suggested the putative catalytic mechanism. The preservation of key active site residues and linker interactions between MetAPs and other enzymes in the same fold suggest that avoiding cross-reactivity can be an important consideration when designing inhibitors that target a single family member.
Structural, biochemical and biological properties
Methionine aminopeptidases (MetAPs) represent a unique class of proteases. Major active site residues and a dinuclear metal center facilitate removal of the N-terminal initiator methionine from nascent polypeptides in a non-procedural manner. Inhibitors against MetAPs offer hope for the treatment of cancer, microbial and fungal infections , , , .
Recently, it was shown that MetAPs are inhibited by the natural product fumarilin and its derivatives. These compounds have been shown to be potent antiangiogenic agents that prevent tumor vascularization and metastasis , . Biochemical and structural evidence has shown that a conserved histidine is the covalent binding site , , , , .
A substrate-type inhibitor based on the natural compound bestatin
The natural substance bestatin has long been known as a potent inhibitor of leucine aminopeptidase (LeuAP) , . Like eMetAP, LeuAP is a dinuclear metalloenzyme, although its general structure is completely different. In the absence of other known inhibitors and in an effort to specifically inhibit eMetAP, the N-terminal side chain of bestatin was replaced by that of norleucine and an alanine was attached to the P1'Item . The resulting non-hydrolyzable
Proposed reaction mechanism
Based on comparisons of the inhibited and native forms of eMetAP, the binding mode of the bestatin-based inhibitor, a kinetic analysis of His-79 and His-178 mutants, and a spectral analysis of the metal center in response to product addition, this possible reaction mechanism was proposed for eMetAP proposed (Fig. 8) , . This mechanism requires: (1) that the N-terminus of the substrate Co2 coordinate at the expense of a terminal solvent molecule; (2) the formation of a
future inhibitor project
Covalent and substrate-like inhibitors of MetAPs serve as a basis for the development of future inhibitors for a variety of pathophysiological conditions. However, the preservation of the active site metal center and flanking histidines will make this task challenging. In an effort to guide experiments examining differences between bacterial, yeast, human, and other "pitta bread" enzymes, a discussion of possible subsite differences is presented.
This work was supported in part by National Research Service Award F32-GM17536 (W.T.L.) and Research Grant GM20066 (B.W.M.) from the National Institutes of Health. Thanks to S. Liu and J. Clardy (Cornell University) for native hMetAP coordinates prior to publication.
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A single amino acid difference between archaeal and human type 2 methionine aminopeptidases distinguishes their affinity for ovalicin
2023, Biochemical and Biophysical Journal – Protein and Proteomics
In almost all living cells, methionine aminopeptidase (MetAP) co-translationally cleaves the methionine primer in at least 70% of newly synthesized polypeptides. MetAPs are commonly classified into Type 1 and Type 2. While prokaryotes and archaea contain only type 1 and type 2 MetAPs, respectively, eukaryotes contain both types of enzymes. Almost all MetAPs published to date only cleave the amino-terminal methionine of substrate peptides. Previous experiments with crude type 2a MetAP isolated fromPyrococci have gone insane(PoohThe MetAP2a cosmid protein library has been shown to cleave leucine in addition to methionine. The authors of this study rejected thePoohMetAP2a activity against leucine substrates and presumably a background reaction contributed by other contaminating proteases. In the current article, using the pure recombinant enzyme, we report that the activity against leucine is actually performed directly by thePoohMetaAP2a. Furthermore, the natural product ovalicin, which is a specific covalent inhibitor of type 2 MetAPs, does not show potent inhibition against thePoohMetaAP2a. Bioinformatic analyzes suggested that a glycine in eukaryotic MetAP2s (G222 in human MetAP2b) and asparagine (N53 inPoohMetAP2a) in archaeal MetAP2s positioned in the analogous position. The N53 side chain forms a hydrogen bond with a conserved histidine (H62) at the entrance to the active site and changes orientation to accommodate ovalicin. This slight difference in the orientation of H62 reduces the affinity of ovalicin by 300,000-fold compared to H62hsMetAP2b inhibition. This difference in activity is partially reduced in the N53G mutationPoohMetAP2a.
Selectfluor-promoted α-methylenation of aromatic ketones to terminal olefins using acetonitrile as the carbon source
2022, letters of the tetrahedron
A method for synthesizing terminal olefins by α-methylenation of aromatic ketones is reported. The reaction was carried out in air using acetonitrile as the carbon source and Selectfluor as a mild oxidant. The scope and versatility of the method were demonstrated using 29 examples. Based on experimental results, an oxidative reaction mechanism promoted by selectfluor is proposed.
Production of pentaglycine fused proteins using the Escherichia coli expression system without in vitro peptidase treatment
2022, Expression and Purification of Proteins
The conjugation of functional molecules to peptides is necessary for protein analysis and applications. Sortase A transpeptidase catalyzes the binding reaction between the LPXTG amino acid sequence and polyglycine and enables specific molecular modifications of the peptide sequence. In this study, the preparation of green fluorescent protein fused to pentaglycine (G5-GFP)vonMethionine shortening mediated byEscherichia coliEndogenous methionylaminopeptidase was examined. Several GFP expression vectors with MetGly5 at the N-terminus were constructed and N-terminal sequence analysis of the expressed protein was performedE coliWe're done. When the first codon of the GFP coding sequence was AUG, a mixture of GFP lacking pentaglycine and G5-GFP was obtained. In contrast, when the first AUG codon was replaced with a codon encoding alanine, G5-GFP was obtained uniformly. These results indicated that the position of AUG in the expression vector had a significant impact on the production of polyglycine fused proteins. The results obtained are useful for the production of casting substrates with polyglycineE coli.
Comparative proteomic analyzes of mycelial, conidial and secreted proteins from Fusarium oxysporum f with high and low pathogenicity. sp. Cucumerinum isolates
2021, Physiological and Molecular Plant Pathology
Fusarium oxysporumF. sp.cucumbers(FOC) is one of the main fungi infecting cucumber plants, causing significant productivity losses. Phytopathogenic fungi vary in disease severity and degree of aggressiveness, with different modes of infection being driven by specialized proteins involved in signaling pathways leading to pathogenicity, an area that is less understood. In order to understand the spectrum of pathogenicity and virulence of FOC isolates, a comprehensive proteomics study was performed to determine the polypeptide patterns of mycelial, conidial and secreted proteins from highly pathogenic (hp-FOC) and low pathogenic (wp-FOC) to compare isolates. Two-dimensional gel electrophoresis (2-DE) coupled with LC-MS/MS analysis led to the identification of common and unique proteins involved in metabolism and pathogenicity in both fungal isolates. This study can describe the biological and molecular basis of different fungal structures to understand the mechanism of their action in host tissues. Determining proteins that express the pathogen's ability to induce pathogenesis can aid in the detection of specialized inhibitors that suppress the activities of pathogen proteins to fight the disease, which can help reduce overuse of fungicides, the still further increasing. plus productivity, quality and environmental safety.
Enzyme | Aminopeptidasen
2021, Encyclopedia of Biological Chemistry: Third Edition
Aminopeptidases, which are widespread in nature, are one of two major subclasses of exopeptidases, proteolytic enzymes that remove amino acids from the ends of peptides and proteins (the others are carboxypeptidases). As the name suggests, aminopeptidases only attack their substrates at the amino-terminal end. Most remove one amino acid at a time, but a small group cleave two or three residues at a time; these are known as dipeptidyl and tripeptidyl aminopeptidases, respectively. Some enzymes such as acylaminoacylpeptidase and pyroglutamylpeptidase remove derived amino acids, but in general aminopeptidases require an unmodified or free amino group.
Discovery of Clioquinol and Analogues as Novel Inhibitors of Severe Acute Respiratory Syndrome Coronavirus-2 Infection, ACE2 and ACE2-In Vitro Spike Protein Interaction
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has led to an ongoing pandemic. There are currently no clinically approved drugs for COVID-19. Therefore, there is an urgent need to accelerate the development of effective antivirals. Here we discover clioquinol (5-chloro-7-iodo-8-quinolinol (CLQ)), a drug approved by the Food and Drug Administration (FDA) and two of its analogues (7-bromo-5-chloro-8-hydroxyquinoline ( CLBQ14) and 5,7-dichloro-8-hydroxyquinoline (CLCQ)) as potent inhibitors of the cytopathic effect induced by SARS-CoV-2 infectionin-vitro.Besides that,All three compounds showed potent anti-exopeptidase activity against recombinant human angiotensin converting enzyme 2 (rhACE2) and inhibited rhACE2 binding to the SARS-CoV-2 spike protein (RBD). CLQ showed the greatest potency in the low micromolar range, with its antiviral activity showing a strong correlation with inhibition of rhACE2 and rhACE2-RBD interaction. Taken together, our results provide a novel mode of action and molecular target for CLQ and validate this pharmacophore as a promising lead series for the clinical development of potential therapeutics for COVID-19.
Featured Articles (6)
The Cys-Arg/N-End regulatory pathway is a general sensor of abiotic stress in flowering plants
Current Biology, Band 27, Heft 20, 2017, S. 3183-3190.e4
Abiotic stress negatively affects plant growth and significantly affects crop yield and quality. While much is known about plant responses, very little is known about initial perceptions of environmental stress at the molecular level. In plants, hypoxia (low oxygen levels that occurs during flooding) is directly mediated by the Cys-Arg/N-terminal pathway of ubiquitin-mediated proteolysis through the oxygen-dependent degradation of group VII ethylene response factor transcription factors (ERFVIIs) via the amino-terminal ( Nt-)cysteine [1,2]. UseArabidopsis(Arabidopsis thaliana) and barley (ordinary barley) we show that the signaling pathway regulates plant responses to multiple abiotic stresses. InArabidopsis, genetic analyzes showed that the response to these stresses is controlled by regulation of the N-end rule ofERFVIIFunction. Oxygen detection via the Cys-Arg/N-End rule in higher eukaryotes is linked to nitric oxide (NO) detection by a unique mechanism [3,4]. In plants, the main mechanism of NO synthesis is via NITRATE REDUCTASE (NR), a nitrogen assimilation enzyme.5]. Here we identified a negative relationship between NR activity and NO levels and the stabilization of an artificial Nt-Cys substrate andERFVIIFunction in response to environmental changes. Also, we show thatERFVIIs amplify abiotic stress responses through physical and genetic interactions with the ATPase BRAHMA, which remodels chromatin. We propose that plants recognize multiple abiotic stresses via the Cys-Arg/N-End pathway, either directly (via oxygen detection) or indirectly (via NO detection after NR activity). This unique mechanism can therefore integrate environment and response to enhance plant survival.
N-terminal protein modifications: bringing back the ribosome
Biochemie, Band 114, 2015, S. 134-146
N-terminal protein modifications correspond to the first modifications that, in principle, any protein can undergo before translation by the ribosome is complete. This class of essential modifications can differ in nature or function and are catalyzed by a variety of dedicated enzymes. Here we give an overview of the current status of the most important N-terminal cotranslational modifications, with a special focus on their catalysts, which belong to the metalloprotease and acyltransferase clans. The first of these modifications corresponds to the excision of the N-terminal methionine, a ubiquitous and essential process leading to the removal of the first methionine. N-alpha acetylation also occurs in all kingdoms, although its extent appears to be significantly increased in higher eukaryotes. Finally, N-myristoylation is a crucial pathway that only exists in eukaryotes. Recent studies on how some of these co-translational modifiers might function in close proximity to the ribosome provide new information on exactly when these modifications occur on the elongating nascent chain and the interaction with other ribosome biogenesis factors responsible for nascent chains are. A comprehensive overview of recent advances in the field of N-terminal protein modifications can be found here.
N-terminal methionine excision of proteins generates tertiary N-degrons that destabilize the Arg/N-tail pathway
Journal of Biological Chemistry, Band 294, Ausgabe 12, 2019, S. 4464-4476
All organisms begin protein synthesis with methionine (Met). The Met primer resulting from the resulting proteins is irreversibly processed by Met aminopeptidases (MetAPs). Excision of the N-terminal (Nt) Met (NME) is an evolutionarily conserved and essential process that acts on up to two-thirds of proteins. However, the universal function of NME is still largely unknown. MetAPs have a known processing preference for Nt-Met with Ala, Ser, Gly, Thr, Cys, Pro, or Val at position 2, but use CHX chase assays to assess protein degradation in yeast cells, as well as protein binding assays and RT-qPCR, we show here, that NME also occurs in nascent proteins that contain Met-Asn or Met-Gln at their N-terminus. We found that NME destabilizes at these tertiary Nt residues (Asn or Gln) of the Arg/N-terminal regulatory pathway that proteins according to the composition of their Nt residues. We also identified a yeast DNA repair protein, MQ-Rad16, bearing a Met-Gln N-terminus, and a human tropomyosin receptor kinase (TFG) gene protein, MN-TFG, bearing a Met-Asn N-terminus as physiological Arg carries /N final rule substrates that are processed by MetAP. Furthermore, we show that the loss of components of the Arg/N-end pathway substantially suppresses the growth defects ofnow 20Δ Yeast cells lacking the catalytic subunit of NatB-Nt acetylase at 37 °C. In summary, the results of our study demonstrate that NME is an important upstream step for the creation of Arg/N-final rule substrates containing tertiary destabilizing residues.live.
Cryo-EM structures show the relocalization of MetAP in the presence of other protein biogenesis factors at the exit of the ribosome tunnel
Journal of Molecular Biology, Band 431, Ausgabe 7, 2019, S. 1426-1439
During protein biosynthesis in bacteria, one of the first events that a nascent polypeptide chain undergoes is enzymatic co-translational processing. The event will involve two enzymatic pathways: N-terminal methionine deformylation by the enzyme peptide deformylase (PDF), followed by methionine excision catalyzed by methionine aminopeptidase (MetAP). During enzymatic processing, the nascent protein is likely protected by the ribosome-associated chaperone trigger. The exit of the ribosomal tunnel serves as a recruitment stage for proteins involved in the maturation processes of the emerging chain. The co-translational processing of nascent chains is a crucial step for the subsequent folding and function of mature proteins.
Here we present cryoelectron microscopic structures ofEscherichia coli(E.coli) ribosome in complex with proteins that process the resulting chain. The structures show overlapping binding sites for PDF and MetAP when individually binding at the tunnel exit site, where the L22-L32 protein region provides primary docking sites for both proteins. In the absence of PDF, the trigger can exit the ribosomal tunnel when MetAP occupies its primary binding site. Interestingly, however, in the presence of PDF, when the primary binding site of MetAP is already activated, MetAP has a remarkable ability to occupy an alternative binding site adjacent to PDF. Our study therefore reveals an unexpected mechanism that MetAP employs for context-specific ribosome association.
Protein N-terminal acetylation: structural basis, mechanism, versatility, and regulation
Trends in Biochemical Sciences, Band 46, Ausgabe 1, 2021, S. 15-27
N-terminal acetylation (NTA) is one of the most common protein modifications, occurring in most eukaryotic proteins but much less common in bacterial and archaeal proteins. This modification is performed by a family of enzymes called N-terminal acetyltransferases (NATs). To date, 12 NATs have been identified, harboring different compositions, substrate specificities, and in some cases modes of regulation. Recent structural and biochemical analyzes of NAT proteins allow comparison of their molecular mechanisms and modes of regulation, which are described here. Although they share an evolutionarily conserved fold and related catalytic mechanism, each catalytic subunit uses unique elements to mediate substrate-specific activity and uses NAT-like specific regulatory and helper subunits for their cellular functions.
Novel reversible purine-based methionine aminopeptidase-2 (MetAP-2) inhibitors and related bicyclic models
Bioorganic & Medicinal Chemistry Letters, Band 27, Ausgabe 3, 2017, S. 551-556
The natural substance fumagillin1and derivatives such as TNP-4702or beloranib3bind irreversibly to methionine aminopeptidase 2 (MetAP-2). This enzyme is crucial for protein maturation and plays a key role in angiogenesis. In this article, we describe the synthesis, binding affinity of MetAP-2, and structural analysis of reversible MetAP-2 inhibitors. The optimization of the enzymatic activity of the screen reached 10 (CI50: 1μM) led to the strongest compound27(CI50: 0.038 μM), with a simultaneous improvement in the LLE from 2.1 to 4.2. Structural analysis of these MetAP-2 inhibitors revealed an unprecedented conformation of the imidazole ring of the His339 side chain, which is coplanar between the imidazole of His331 and the aryl ether moiety attached to the purine backbone. Systematic modification and reduction of the H-bonding capacity of this metal-binding moiety induced an unexpected 180° rotation of the triazolo[1,5-A] bicyclic mold of pyrimidine.
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