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1.
ChemMedChem ; 2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34331400

RESUMO

Metallohydrolases form a large group of enzymes that have fundamental importance in a broad range of biological functions. Among them, the purple acid phosphatases (PAPs) have gained attention due to their crucial role in the acquisition and use of phosphate by plants and also as a promising target for novel treatments of bone-related disorders and cancer. To date, no crystal structure of a mammalian PAP with drug-like molecules bound near the active site is available. Herein, we used a fragment-based design approach using structures of a mammalian PAP in complex with the MaybridgeTM fragment CC063346, the amino acid L-glutamine and the buffer molecule HEPES, as well as various solvent molecules to guide the design of highly potent and efficient mammalian PAP inhibitors. These inhibitors have improved aqueous solubility when compared to the clinically most promising PAP inhibitors available to date. Furthermore, drug-like fragments bound in newly discovered binding sites mapped out additional scaffolds for further inhibitor discovery, as well as scaffolds for the design of inhibitors with novel modes of action.

2.
Nat Commun ; 12(1): 4621, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34330928

RESUMO

Cytochromes bd are ubiquitous amongst prokaryotes including many human-pathogenic bacteria. Such complexes are targets for the development of antimicrobial drugs. However, an understanding of the relationship between the structure and functional mechanisms of these oxidases is incomplete. Here, we have determined the 2.8 Å structure of Mycobacterium smegmatis cytochrome bd by single-particle cryo-electron microscopy. This bd oxidase consists of two subunits CydA and CydB, that adopt a pseudo two-fold symmetrical arrangement. The structural topology of its Q-loop domain, whose function is to bind the substrate, quinol, is significantly different compared to the C-terminal region reported for cytochromes bd from Geobacillus thermodenitrificans (G. th) and Escherichia coli (E. coli). In addition, we have identified two potential oxygen access channels in the structure and shown that similar tunnels also exist in G. th and E. coli cytochromes bd. This study provides insights to develop a framework for the rational design of antituberculosis compounds that block the oxygen access channels of this oxidase.


Assuntos
Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica/métodos , Grupo dos Citocromos b/ultraestrutura , Complexo de Proteínas da Cadeia de Transporte de Elétrons/ultraestrutura , Mycobacterium smegmatis/enzimologia , Oxirredutases/ultraestrutura , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Grupo dos Citocromos b/química , Grupo dos Citocromos b/metabolismo , Transporte de Elétrons , Complexo de Proteínas da Cadeia de Transporte de Elétrons/química , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Heme/química , Heme/metabolismo , Modelos Moleculares , Mycobacterium smegmatis/genética , Oxirredutases/química , Oxirredutases/metabolismo , Oxigênio/metabolismo , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Especificidade por Substrato
3.
Cell ; 184(13): 3474-3485.e11, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34143953

RESUMO

The capping of mRNA and the proofreading play essential roles in SARS-CoV-2 replication and transcription. Here, we present the cryo-EM structure of the SARS-CoV-2 replication-transcription complex (RTC) in a form identified as Cap(0)-RTC, which couples a co-transcriptional capping complex (CCC) composed of nsp12 NiRAN, nsp9, the bifunctional nsp14 possessing an N-terminal exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase), and nsp10 as a cofactor of nsp14. Nsp9 and nsp12 NiRAN recruit nsp10/nsp14 into the Cap(0)-RTC, forming the N7-CCC to yield cap(0) (7MeGpppA) at 5' end of pre-mRNA. A dimeric form of Cap(0)-RTC observed by cryo-EM suggests an in trans backtracking mechanism for nsp14 ExoN to facilitate proofreading of the RNA in concert with polymerase nsp12. These results not only provide a structural basis for understanding co-transcriptional modification of SARS-CoV-2 mRNA but also shed light on how replication fidelity in SARS-CoV-2 is maintained.


Assuntos
RNA-Polimerase RNA-Dependente de Coronavírus/genética , Exorribonucleases/genética , Metiltransferases/genética , SARS-CoV-2/genética , Sequência de Aminoácidos , COVID-19/virologia , Humanos , RNA Mensageiro/genética , RNA Viral/genética , Alinhamento de Sequência , Transcrição Genética/genética , Replicação Viral/genética
4.
Eur J Med Chem ; 219: 113416, 2021 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-33887682

RESUMO

Parasites of the Plasmodium genus are unable to produce purine nucleotides de novo and depend completely on the salvage pathway. This fact makes plasmodial hypoxanthine-guanine-(xanthine) phosphoribosyltransferase [HG(X)PRT] a valuable target for development of antimalarial agents. A series of nucleotide analogues was designed, synthesized and evaluated as potential inhibitors of Plasmodium falciparum HGXPRT, P. vivax HGPRT and human HGPRT. These novel nucleoside phosphonates have a pyrrolidine, piperidine or piperazine ring incorporated into the linker connecting the purine base to a phosphonate group(s) and exhibited a broad range of Ki values between 0.15 and 72 µM. The corresponding phosphoramidate prodrugs, able to cross cell membranes, have been synthesized and evaluated in a P. falciparum infected human erythrocyte assay. Of the eight prodrugs evaluated seven exhibited in vitro antimalarial activity with IC50 values within the range of 2.5-12.1 µM. The bis-phosphoramidate prodrug 13a with a mean (SD) IC50 of 2.5 ± 0.7 µM against the chloroquine-resistant P. falciparum W2 strain exhibited low cytotoxicity in the human hepatocellular liver carcinoma (HepG2) and normal human dermal fibroblasts (NHDF) cell lines at a concentration of 100 µM suggesting good selectivity for further structure-activity relationship investigations.


Assuntos
Antimaláricos/síntese química , Inibidores Enzimáticos/química , Nucleotídeos/química , Pentosiltransferases/antagonistas & inibidores , Proteínas de Protozoários/antagonistas & inibidores , Antimaláricos/metabolismo , Antimaláricos/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Resistência a Medicamentos/efeitos dos fármacos , Inibidores Enzimáticos/metabolismo , Eritrócitos/citologia , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Humanos , Nucleotídeos/metabolismo , Pentosiltransferases/metabolismo , Piperazina/química , Piperidinas/química , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Plasmodium vivax/enzimologia , Pró-Fármacos/síntese química , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacologia , Proteínas de Protozoários/metabolismo , Pirrolidinas/química , Relação Estrutura-Atividade
5.
Proc Natl Acad Sci U S A ; 118(15)2021 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-33876763

RESUMO

Complex II, also known as succinate dehydrogenase (SQR) or fumarate reductase (QFR), is an enzyme involved in both the Krebs cycle and oxidative phosphorylation. Mycobacterial Sdh1 has recently been identified as a new class of respiratory complex II (type F) but with an unknown electron transfer mechanism. Here, using cryoelectron microscopy, we have determined the structure of Mycobacterium smegmatis Sdh1 in the presence and absence of the substrate, ubiquinone-1, at 2.53-Å and 2.88-Å resolution, respectively. Sdh1 comprises three subunits, two that are water soluble, SdhA and SdhB, and one that is membrane spanning, SdhC. Within these subunits we identified a quinone-binding site and a rarely observed Rieske-type [2Fe-2S] cluster, the latter being embedded in the transmembrane region. A mutant, where two His ligands of the Rieske-type [2Fe-2S] were changed to alanine, abolished the quinone reduction activity of the Sdh1. Our structures allow the proposal of an electron transfer pathway that connects the substrate-binding and quinone-binding sites. Given the unique features of Sdh1 and its essential role in Mycobacteria, these structures will facilitate antituberculosis drug discovery efforts that specifically target this complex.

6.
J Med Chem ; 64(9): 5710-5729, 2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-33891818

RESUMO

Helicobacter pylori (Hp) is a human pathogen that lives in the gastric mucosa of approximately 50% of the world's population causing gastritis, peptic ulcers, and gastric cancer. An increase in resistance to current drugs has sparked the search for new Hp drug targets and therapeutics. One target is the disruption of nucleic acid production, which can be achieved by impeding the synthesis of 6-oxopurine nucleoside monophosphates, the precursors of DNA and RNA. These metabolites are synthesized by Hp xanthine-guanine-hypoxanthine phosphoribosyltransferase (XGHPRT). Here, nucleoside phosphonates have been evaluated, which inhibit the activity of this enzyme with Ki values as low as 200 nM. The prodrugs of these compounds arrest the growth of Hp at a concentration of 50 µM in cell-based assays. The kinetic properties of HpXGHPRT have been determined together with its X-ray crystal structure in the absence and presence of 9-[(N-3-phosphonopropyl)-aminomethyl-9-deazahypoxanthine, providing a basis for new antibiotic development.


Assuntos
Antibacterianos/química , Proteínas de Bactérias/metabolismo , Pentosiltransferases/metabolismo , Sequência de Aminoácidos , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Proteínas de Bactérias/química , Sítios de Ligação , Cristalografia por Raios X , Gastroenteropatias/tratamento farmacológico , Gastroenteropatias/microbiologia , Gastroenteropatias/patologia , Infecções por Helicobacter/tratamento farmacológico , Infecções por Helicobacter/patologia , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/enzimologia , Humanos , Hipoxantina Fosforribosiltransferase/química , Hipoxantina Fosforribosiltransferase/metabolismo , Hipoxantinas/química , Hipoxantinas/metabolismo , Hipoxantinas/farmacologia , Hipoxantinas/uso terapêutico , Cinética , Simulação de Dinâmica Molecular , Organofosfonatos/química , Organofosfonatos/metabolismo , Organofosfonatos/farmacologia , Organofosfonatos/uso terapêutico , Pentosiltransferases/química , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacologia , Pró-Fármacos/uso terapêutico , Alinhamento de Sequência , Relação Estrutura-Atividade
7.
Protein Cell ; 2021 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-33864621

RESUMO

A new coronavirus (SARS-CoV-2) has been identified as the etiologic agent for the COVID-19 outbreak. Currently, effective treatment options remain very limited for this disease; therefore, there is an urgent need to identify new anti-COVID-19 agents. In this study, we screened over 6,000 compounds that included approved drugs, drug candidates in clinical trials, and pharmacologically active compounds to identify leads that target the SARS-CoV-2 papain-like protease (PLpro). Together with main protease (Mpro), PLpro is responsible for processing the viral replicase polyprotein into functional units. Therefore, it is an attractive target for antiviral drug development. Here we discovered four compounds, YM155, cryptotanshinone, tanshinone I and GRL0617 that inhibit SARS-CoV-2 PLpro with IC50 values ranging from 1.39 to 5.63 µmol/L. These compounds also exhibit strong antiviral activities in cell-based assays. YM155, an anticancer drug candidate in clinical trials, has the most potent antiviral activity with an EC50 value of 170 nmol/L. In addition, we have determined the crystal structures of this enzyme and its complex with YM155, revealing a unique binding mode. YM155 simultaneously targets three "hot" spots on PLpro, including the substrate-binding pocket, the interferon stimulating gene product 15 (ISG15) binding site and zinc finger motif. Our results demonstrate the efficacy of this screening and repurposing strategy, which has led to the discovery of new drug leads with clinical potential for COVID-19 treatments.

8.
J Med Chem ; 64(3): 1670-1684, 2021 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-33512163

RESUMO

New drugs to treat tuberculosis (TB) are urgently needed to combat the increase in resistance observed among the current first-line and second-line treatments. Here, we propose ketol-acid reductoisomerase (KARI) as a target for anti-TB drug discovery. Twenty-two analogues of IpOHA, an inhibitor of plant KARI, were evaluated as antimycobacterial agents. The strongest inhibitor of Mycobacterium tuberculosis (Mt) KARI has a Ki value of 19.7 nM, fivefold more potent than IpOHA (Ki = 97.7 nM). This and four other potent analogues are slow- and tight-binding inhibitors of MtKARI. Three compounds were cocrystallized with Staphylococcus aureus KARI and yielded crystals that diffracted to 1.6-2.0 Å resolution. Prodrugs of these compounds possess antimycobacterial activity against H37Rv, a virulent strain of human TB, with the most active compound having an MIC90 of 2.32 ± 0.04 µM. This compound demonstrates a very favorable selectivity window and represents a highly promising lead as an anti-TB agent.


Assuntos
Antituberculosos/farmacologia , Herbicidas/farmacologia , Cetol-Ácido Redutoisomerase/antagonistas & inibidores , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Animais , Antituberculosos/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Descoberta de Drogas , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Simulação de Acoplamento Molecular , Pró-Fármacos , Staphylococcus aureus/enzimologia
9.
Cell ; 184(1): 184-193.e10, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-33232691

RESUMO

Transcription of SARS-CoV-2 mRNA requires sequential reactions facilitated by the replication and transcription complex (RTC). Here, we present a structural snapshot of SARS-CoV-2 RTC as it transitions toward cap structure synthesis. We determine the atomic cryo-EM structure of an extended RTC assembled by nsp7-nsp82-nsp12-nsp132-RNA and a single RNA-binding protein, nsp9. Nsp9 binds tightly to nsp12 (RdRp) NiRAN, allowing nsp9 N terminus inserting into the catalytic center of nsp12 NiRAN, which then inhibits activity. We also show that nsp12 NiRAN possesses guanylyltransferase activity, catalyzing the formation of cap core structure (GpppA). The orientation of nsp13 that anchors the 5' extension of template RNA shows a remarkable conformational shift, resulting in zinc finger 3 of its ZBD inserting into a minor groove of paired template-primer RNA. These results reason an intermediate state of RTC toward mRNA synthesis, pave a way to understand the RTC architecture, and provide a target for antiviral development.


Assuntos
RNA-Polimerase RNA-Dependente de Coronavírus/química , Microscopia Crioeletrônica , RNA Mensageiro/química , RNA Viral/química , SARS-CoV-2/química , Proteínas do Complexo da Replicase Viral/química , Sequência de Aminoácidos , Coronavirus/química , Coronavirus/classificação , Coronavirus/enzimologia , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Metiltransferases/metabolismo , Modelos Moleculares , RNA Helicases/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , SARS-CoV-2/enzimologia , Alinhamento de Sequência , Transcrição Genética , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Replicação Viral
10.
Chemistry ; 27(9): 3130-3141, 2021 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-33215746

RESUMO

New drugs aimed at novel targets are urgently needed to combat the increasing rate of drug-resistant tuberculosis (TB). Herein, the National Cancer Institute Developmental Therapeutic Program (NCI-DTP) chemical library was screened against a promising new target, ketol-acid reductoisomerase (KARI), the second enzyme in the branched-chain amino acid (BCAA) biosynthesis pathway. From this library, 6-hydroxy-2-methylthiazolo[4,5-d]pyrimidine-5,7(4H,6H)-dione (NSC116565) was identified as a potent time-dependent inhibitor of Mycobacterium tuberculosis (Mt) KARI with a Ki of 95.4 nm. Isothermal titration calorimetry studies showed that this inhibitor bound to MtKARI in the presence and absence of the cofactor, nicotinamide adenine dinucleotide phosphate (NADPH), which was confirmed by crystal structures of the compound in complex with closely related Staphylococcus aureus KARI. It is also shown that NSC116565 inhibits the growth of H37Ra and H37Rv strains of Mt with MIC50 values of 2.93 and 6.06 µm, respectively. These results further validate KARI as a TB drug target and show that NSC116565 is a promising lead for anti-TB drug development.


Assuntos
Antituberculosos/farmacologia , Cetol-Ácido Redutoisomerase/antagonistas & inibidores , Mycobacterium tuberculosis/enzimologia , Pirimidinonas/farmacologia , Linhagem Celular , Humanos , Cetol-Ácido Redutoisomerase/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , NADP/metabolismo , Staphylococcus aureus/enzimologia , Tuberculose/tratamento farmacológico , Tuberculose/microbiologia
11.
Sci Adv ; 6(44)2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33127676

RESUMO

In bacteria, adenosine 5'-triphosphate (ATP)-binding cassette (ABC) importers are essential for the uptake of nutrients including the nonreducing disaccharide trehalose, a metabolite that is crucial for the survival and virulence of several human pathogens including Mycobacterium tuberculosis SugABC is an ABC transporter that translocates trehalose from the periplasmic lipoprotein LpqY into the cytoplasm of mycobacteria. Here, we report four high-resolution cryo-electron microscopy structures of the mycobacterial LpqY-SugABC complex to reveal how it binds and passes trehalose through the membrane to the cytoplasm. A unique feature observed in this system is the initial mode of capture of the trehalose at the LpqY interface. Uptake is achieved by a pivotal rotation of LpqY relative to SugABC, moving from an open and accessible conformation to a clamped conformation upon trehalose binding. These findings enrich our understanding as to how ABC transporters facilitate substrate transport across the membrane in Gram-positive bacteria.

12.
ACS Infect Dis ; 6(11): 2901-2912, 2020 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-32986949

RESUMO

Acetohydroxyacid synthase (AHAS, EC 2.2.1.6), the first enzyme in the branched chain amino acid biosynthesis pathway, is the target for more than 50 commercially available herbicides, and is a promising target for antimicrobial drug discovery. Herein, we have expressed and purified AHAS from Candida auris, a newly identified human invasive fungal pathogen. Thirteen AHAS inhibiting herbicides have Ki values of <2 µM for this enzyme, with the most potent having Ki values of <32 nM. Six of these compounds exhibited MIC50 values of <1 µM against C. auris (CBS10913 strain) grown in culture, with bensulfuron methyl (BSM) being fungicidal and the most potent (MIC50 of 0.090 µM) in defined minimal media. The MIC50 value increases to 0.90 µM in media enriched by the addition of branched-chain amino acids at the expected concentration in the blood serum. The sessile MIC50 for BSM is 0.6 µM. Thus, it is also an excellent inhibitor of the growth of C. auris biofilms. BSM is nontoxic in HEK-293 cells at concentrations >100 µM and thus possesses a therapeutic index of >100. These data suggest that targeting AHAS is a viable strategy for treating C. auris infections.


Assuntos
Acetolactato Sintase , Herbicidas , Preparações Farmacêuticas , Acetolactato Sintase/genética , Candida , Células HEK293 , Humanos
13.
Nat Commun ; 11(1): 4245, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32843629

RESUMO

Diheme-containing succinate:menaquinone oxidoreductases (Sdh) are widespread in Gram-positive bacteria but little is known about the catalytic mechanisms they employ for succinate oxidation by menaquinone. Here, we present the 2.8 Å cryo-electron microscopy structure of a Mycobacterium smegmatis Sdh, which forms a trimer. We identified the membrane-anchored SdhF as a subunit of the complex. The 3 kDa SdhF forms a single transmembrane helix and this helix plays a role in blocking the canonically proximal quinone-binding site. We also identified two distal quinone-binding sites with bound quinones. One distal binding site is formed by neighboring subunits of the complex. Our structure further reveals the electron/proton transfer pathway for succinate oxidation by menaquinone. Moreover, this study provides further structural insights into the physiological significance of a trimeric respiratory complex II. The structure of the menaquinone binding site could provide a framework for the development of Sdh-selective anti-mycobacterial drugs.


Assuntos
Proteínas de Bactérias/química , Mycobacterium smegmatis/enzimologia , Succinato Desidrogenase/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Catálise , Microscopia Crioeletrônica , Transporte de Elétrons , Modelos Moleculares , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Mycobacterium smegmatis/química , Oxirredução , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Relação Estrutura-Atividade , Succinato Desidrogenase/metabolismo , Ácido Succínico/metabolismo , Vitamina K 2/metabolismo
14.
Arch Biochem Biophys ; 692: 108516, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32745463

RESUMO

Ketol-acid reductoisomerase (KARI), the second enzyme in the branched-chain amino acid (BCAA) biosynthesis pathway, is an emerging target for the discovery of biocides. Here, we demonstrate that cyclopropane-1,1-dicarboxylate (CPD) inhibits KARIs from the pathogens Mycobacterium tuberculosis (Mt) and Campylobacter jejuni (Cj) reversibly with Ki values of 3.03 µM and 0.59 µM, respectively. Another reversible inhibitor of both KARIs, Hoe 704, is more potent than CPD with Ki values of 300 nM and 110 nM for MtKARI and CjKARI, respectively. The most potent inhibitor tested here is N-hydroxy-N-isopropyloxamate (IpOHA). It has a Ki of ~26 nM for MtKARI, but binds rather slowly (kon ~900 M-1s-1). In contrast, IpOHA binds more rapidly (kon ~7000 M-1s-1) to CjKARI and irreversibly.


Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Campylobacter jejuni/enzimologia , Inibidores Enzimáticos/química , Cetol-Ácido Redutoisomerase/antagonistas & inibidores , Mycobacterium tuberculosis/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Campylobacter jejuni/química , Ciclopropanos/química , Ácidos Dicarboxílicos/química , Ácidos Hidroxâmicos/química , Cetol-Ácido Redutoisomerase/química , Cetol-Ácido Redutoisomerase/metabolismo , Mycobacterium tuberculosis/química , Compostos Organofosforados/química
15.
Sci Rep ; 10(1): 12882, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32732933

RESUMO

Genes that confer antibiotic resistance can rapidly be disseminated from one microorganism to another by mobile genetic elements, thus transferring resistance to previously susceptible bacterial strains. The misuse of antibiotics in health care and agriculture has provided a powerful evolutionary pressure to accelerate the spread of resistance genes, including those encoding ß-lactamases. These are enzymes that are highly efficient in inactivating most of the commonly used ß-lactam antibiotics. However, genes that confer antibiotic resistance are not only associated with pathogenic microorganisms, but are also found in non-pathogenic (i.e. environmental) microorganisms. Two recent examples are metal-dependent ß-lactamases (MBLs) from the marine organisms Novosphingobium pentaromativorans and Simiduia agarivorans. Previous studies have demonstrated that their ß-lactamase activity is comparable to those of well-known MBLs from pathogenic sources (e.g. NDM-1, AIM-1) but that they also possess efficient lactonase activity, an activity associated with quorum sensing. Here, we probed the structure and mechanism of these two enzymes using crystallographic, spectroscopic and fast kinetics techniques. Despite highly conserved active sites both enzymes demonstrate significant variations in their reaction mechanisms, highlighting both the extraordinary ability of MBLs to adapt to changing environmental conditions and the rather promiscuous acceptance of diverse substrates by these enzymes.


Assuntos
Organismos Aquáticos/enzimologia , Proteínas de Bactérias/química , Gammaproteobacteria/enzimologia , Sphingomonadaceae/enzimologia , beta-Lactamases/química , 4-Butirolactona/análogos & derivados , 4-Butirolactona/química , 4-Butirolactona/metabolismo , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , beta-Lactamases/metabolismo , beta-Lactamas/química , beta-Lactamas/metabolismo
16.
Nature ; 586(7828): 317-321, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32640464

RESUMO

Acetohydroxyacid synthase (AHAS), also known as acetolactate synthase, is a flavin adenine dinucleotide-, thiamine diphosphate- and magnesium-dependent enzyme that catalyses the first step in the biosynthesis of branched-chain amino acids1. It is the target for more than 50 commercial herbicides2. AHAS requires both catalytic and regulatory subunits for maximal activity and functionality. Here we describe structures of the hexadecameric AHAS complexes of Saccharomyces cerevisiae and dodecameric AHAS complexes of Arabidopsis thaliana. We found that the regulatory subunits of these AHAS complexes form a core to which the catalytic subunit dimers are attached, adopting the shape of a Maltese cross. The structures show how the catalytic and regulatory subunits communicate with each other to provide a pathway for activation and for feedback inhibition by branched-chain amino acids. We also show that the AHAS complex of Mycobacterium tuberculosis adopts a similar structure, thus demonstrating that the overall AHAS architecture is conserved across kingdoms.


Assuntos
Acetolactato Sintase/química , Arabidopsis/enzimologia , Saccharomyces cerevisiae/enzimologia , Acetolactato Sintase/metabolismo , Trifosfato de Adenosina/metabolismo , Aminoácidos de Cadeia Ramificada/biossíntese , Domínio Catalítico , Ativação Enzimática , Evolução Molecular , Retroalimentação Fisiológica , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Mycobacterium tuberculosis/enzimologia , Ligação Proteica , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Valina/metabolismo
17.
Proc Natl Acad Sci U S A ; 117(28): 16324-16332, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601219

RESUMO

FadE, an acyl-CoA dehydrogenase, introduces unsaturation to carbon chains in lipid metabolism pathways. Here, we report that FadE5 from Mycobacterium tuberculosis (MtbFadE5) and Mycobacterium smegmatis (MsFadE5) play roles in drug resistance and exhibit broad specificity for linear acyl-CoA substrates but have a preference for those with long carbon chains. Here, the structures of MsFadE5 and MtbFadE5, in the presence and absence of substrates, have been determined. These reveal the molecular basis for the broad substrate specificity of these enzymes. FadE5 interacts with the CoA region of the substrate through a large number of hydrogen bonds and an unusual π-π stacking interaction, allowing these enzymes to accept both short- and long-chain substrates. Residues in the substrate binding cavity reorient their side chains to accommodate substrates of various lengths. Longer carbon-chain substrates make more numerous hydrophobic interactions with the enzyme compared with the shorter-chain substrates, resulting in a preference for this type of substrate.


Assuntos
Acil-CoA Desidrogenase/química , Acil-CoA Desidrogenase/metabolismo , Mycobacterium/enzimologia , Acil Coenzima A/metabolismo , Acil-CoA Desidrogenase/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Domínio Catalítico , Farmacorresistência Bacteriana/genética , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Modelos Moleculares , Mutação , Mycobacterium/efeitos dos fármacos , Mycobacterium/genética , Conformação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato
18.
Cell ; 182(2): 417-428.e13, 2020 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-32526208

RESUMO

Nucleotide analog inhibitors, including broad-spectrum remdesivir and favipiravir, have shown promise in in vitro assays and some clinical studies for COVID-19 treatment, this despite an incomplete mechanistic understanding of the viral RNA-dependent RNA polymerase nsp12 drug interactions. Here, we examine the molecular basis of SARS-CoV-2 RNA replication by determining the cryo-EM structures of the stalled pre- and post- translocated polymerase complexes. Compared with the apo complex, the structures show notable structural rearrangements happening to nsp12 and its co-factors nsp7 and nsp8 to accommodate the nucleic acid, whereas there are highly conserved residues in nsp12, positioning the template and primer for an in-line attack on the incoming nucleotide. Furthermore, we investigate the inhibition mechanism of the triphosphate metabolite of remdesivir through structural and kinetic analyses. A transition model from the nsp7-nsp8 hexadecameric primase complex to the nsp12-nsp7-nsp8 polymerase complex is also proposed to provide clues for the understanding of the coronavirus transcription and replication machinery.


Assuntos
Betacoronavirus/química , Betacoronavirus/enzimologia , RNA Polimerase Dependente de RNA/química , Proteínas não Estruturais Virais/química , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/química , Alanina/metabolismo , Alanina/farmacologia , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacologia , Domínio Catalítico , RNA-Polimerase RNA-Dependente de Coronavírus , Microscopia Crioeletrônica , Modelos Químicos , Modelos Moleculares , RNA Viral/metabolismo , SARS-CoV-2 , Transcrição Genética , Replicação Viral
19.
J Mol Biol ; 432(16): 4426-4434, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32512002

RESUMO

Novel antitubercular agents are urgently needed to combat the emergence of global drug resistance to human tuberculosis. Mycobacterial membrane protein Large 3 (MmpL3) is a promising drug target because its activity is essential and required for cell-wall biosynthesis. Several classes of MmpL3 inhibitors have been developed against Mycobacterium tuberculosis (Mtb) with potent anti-tuberculosis activity. These include the drug candidate SQ109, which has progressed to phase IIb/III clinical trials. Here, we have determined the crystal structures of MmpL3 in complex with NITD-349 and SPIRO. Both inhibitors bind deep in the central channel of transmembrane domain and cause conformational changes to the protein. The amide nitrogen and indole nitrogen of NITD-349 and the piperidine nitrogen of SPIRO interact and clamp Asp645. Structural analysis of the two structures reveals that these inhibitors target the proton relay pathway to block the activity of MmpL3. The findings presented here enrich our understanding of the binding modes of MmpL3 inhibitors and provide directions to enable further rational drug design targeting MmpL3.


Assuntos
Antituberculosos/farmacologia , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Mycobacterium smegmatis/metabolismo , Antituberculosos/química , Sítios de Ligação , Cristalografia por Raios X , Indóis/química , Indóis/farmacologia , Modelos Moleculares , Mycobacterium smegmatis/efeitos dos fármacos , Piperidinas/química , Piperidinas/farmacologia , Ligação Proteica , Compostos de Espiro/química , Compostos de Espiro/farmacologia
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