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1.
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
2.
Sci Rep ; 11(1): 12410, 2021 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-34127732

RESUMO

In situ generation of antibacterial and antiviral agents by harnessing the catalytic activity of enzymes on surfaces provides an effective eco-friendly approach for disinfection. The perhydrolase (AcT) from Mycobacterium smegmatis catalyzes the perhydrolysis of acetate esters to generate the potent disinfectant, peracetic acid (PAA). In the presence of AcT and its two substrates, propylene glycol diacetate and H2O2, sufficient and continuous PAA is generated over an extended time to kill a wide range of bacteria with the enzyme dissolved in aqueous buffer. For extended self-disinfection, however, active and stable AcT bound onto or incorporated into a surface coating is necessary. In the current study, an active, stable and reusable AcT-based coating was developed by incorporating AcT into a polydopamine (PDA) matrix in a single step, thereby forming a biocatalytic composite onto a variety of surfaces. The resulting AcT-PDA composite coatings on glass, metal and epoxy surfaces yielded up to 7-log reduction of Gram-positive and Gram-negative bacteria when in contact with the biocatalytic coating. This composite coating also possessed potent antiviral activity, and dramatically reduced the infectivity of a SARS-CoV-2 pseudovirus within minutes. The single-step approach enables rapid and facile fabrication of enzyme-based disinfectant composite coatings with high activity and stability, which enables reuse following surface washing. As a result, this enzyme-polymer composite technique may serve as a general strategy for preparing antibacterial and antiviral surfaces for applications in health care and common infrastructure safety, such as in schools, the workplace, transportation, etc.


Assuntos
Antibacterianos/química , Antivirais/química , Proteínas de Bactérias/química , Hidrolases/química , Indóis/química , Polímeros/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Antivirais/metabolismo , Antivirais/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , COVID-19/patologia , COVID-19/virologia , Materiais Revestidos Biocompatíveis/química , Materiais Revestidos Biocompatíveis/metabolismo , Materiais Revestidos Biocompatíveis/farmacologia , Estabilidade de Medicamentos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Humanos , Hidrolases/genética , Hidrolases/metabolismo , Cinética , Mycobacterium smegmatis/enzimologia , Ácido Peracético/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , SARS-CoV-2/efeitos dos fármacos
3.
Commun Biol ; 4(1): 684, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083757

RESUMO

Glutamate dehydrogenases (GDHs) are widespread metabolic enzymes that play key roles in nitrogen homeostasis. Large glutamate dehydrogenases composed of 180 kDa subunits (L-GDHs180) contain long N- and C-terminal segments flanking the catalytic core. Despite the relevance of L-GDHs180 in bacterial physiology, the lack of structural data for these enzymes has limited the progress of functional studies. Here we show that the mycobacterial L-GDH180 (mL-GDH180) adopts a quaternary structure that is radically different from that of related low molecular weight enzymes. Intersubunit contacts in mL-GDH180 involve a C-terminal domain that we propose as a new fold and a flexible N-terminal segment comprising ACT-like and PAS-type domains that could act as metabolic sensors for allosteric regulation. These findings uncover unique aspects of the structure-function relationship in the subfamily of L-GDHs.


Assuntos
Proteínas de Bactérias/química , Glutamato Desidrogenase/química , Mycobacterium smegmatis/enzimologia , Proteínas Recombinantes/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Microscopia Crioeletrônica , Cristalografia por Raios X , Glutamato Desidrogenase/metabolismo , Glutamato Desidrogenase/ultraestrutura , Cinética , Modelos Moleculares , Mycobacterium smegmatis/genética , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
4.
Biochem Biophys Res Commun ; 560: 159-164, 2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-33992958

RESUMO

The Kemp elimination reaction, involving the ring-opening of benzoxazole and its derivatives under the action of natural enzymes or chemical catalysts, has been of interest to researchers since its discovery. Because this reaction does not exist in all currently known metabolic pathways, the computational design of Kemp eliminases has provided valuable insights into principles of enzymatic catalysis. However, it was discovered that the naturally occurring promiscuous enzymes ydbC, xapA and ketosteroid isomerase also can catalyze Kemp elimination. Here, we report the crystal structure of ketosteroid isomerase (KSI) from Mycobacterium smegmatis MC2 155. MsKSI crystallizes in the P212121 space group with two molecules in an asymmetric unit, and ultracentrifugation data confirms that it forms a stable dimer in solution, consistent with the 1.9 Å-resolution structure. Our assays confirm that MsKSI accelerates the Kemp elimination of 5-nitrobenzoxazole (5NBI) with an optimal pH of 5.5. A 2.35 Å resolution crystal structure of the MsKSI-5NBI complex reveals that the substrate 5NBI is bound in the active pocket of the enzyme composed of hydrophobic residues. In addition, the Glu127 residue is proposed to play an important role as a general base in proton transfer and breaking weak O-N bonds to open the five-membered ring. This work provides a starting point for exploring the artificial modification of MsKSI using the natural enzyme as the backbone.


Assuntos
Proteínas de Bactérias/química , Mycobacterium smegmatis/enzimologia , Esteroide Isomerases/química , Proteínas de Bactérias/metabolismo , Biocatálise , Cristalografia por Raios X , Modelos Moleculares , Subunidades Proteicas/química , Esteroide Isomerases/metabolismo
5.
Int J Mol Sci ; 22(8)2021 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-33918623

RESUMO

Compounds targeting bacterial topoisomerases are of interest for the development of antibacterial agents. Our previous studies culminated in the synthesis and characterization of small-molecular weight thiosemicarbazides as the initial prototypes of a novel class of gyrase and topoisomerase IV inhibitors. To expand these findings with further details on the mode of action of the most potent compounds, enzymatic studies combined with a molecular docking approach were carried out, the results of which are presented herein. The biochemical assay for 1-(indol-2-oyl)-4-(4-nitrophenyl) thiosemicarbazide (4) and 4-benzoyl-1-(indol-2-oyl) thiosemicarbazide (7), showing strong inhibitory activity against Staphylococcus aureus topoisomerase IV, confirmed that these compounds reduce the ability of the ParE subunit to hydrolyze ATP rather than act by stabilizing the cleavage complex. Compound 7 showed better antibacterial activity than compound 4 against clinical strains of S. aureus and representatives of the Mycobacterium genus. In vivo studies using time-lapse microfluidic microscopy, which allowed for the monitoring of fluorescently labelled replisomes, revealed that compound 7 caused an extension of the replication process duration in Mycobacterium smegmatis, as well as the growth arrest of bacterial cells. Despite some similarities to the mechanism of action of novobiocin, these compounds show additional, unique properties, and can thus be considered a novel group of inhibitors of the ATPase activity of bacterial type IIA topoisomerases.


Assuntos
Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/enzimologia , Semicarbazidas/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/enzimologia , Antibacterianos/química , Sítios de Ligação , DNA Girase/química , Inibidores Enzimáticos/química , Humanos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Semicarbazidas/química , Relação Estrutura-Atividade , Inibidores da Topoisomerase/química , Inibidores da Topoisomerase/farmacologia
6.
Nucleic Acids Res ; 49(7): 3876-3887, 2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33744958

RESUMO

Mycobacterium smegmatis Lhr (MsmLhr; 1507-aa) is the founder of a novel clade of bacterial helicases. MsmLhr consists of an N-terminal helicase domain (aa 1-856) with a distinctive tertiary structure (Lhr-Core) and a C-terminal domain (Lhr-CTD) of unknown structure. Here, we report that Escherichia coli Lhr (EcoLhr; 1538-aa) is an ATPase, translocase and ATP-dependent helicase. Like MsmLhr, EcoLhr translocates 3' to 5' on ssDNA and unwinds secondary structures en route, with RNA:DNA hybrid being preferred versus DNA:DNA duplex. The ATPase and translocase activities of EcoLhr inhere to its 877-aa Core domain. Full-length EcoLhr and MsmLhr have homo-oligomeric quaternary structures in solution, whereas their respective Core domains are monomers. The MsmLhr CTD per se is a homo-oligomer in solution. We employed cryo-EM to solve the structure of the CTD of full-length MsmLhr. The CTD protomer is composed of a series of five winged-helix (WH) modules and a ß-barrel module. The CTD adopts a unique homo-tetrameric quaternary structure. A Lhr-CTD subdomain, comprising three tandem WH modules and the ß-barrel, is structurally homologous to AlkZ, a bacterial DNA glycosylase that recognizes and excises inter-strand DNA crosslinks. This homology is noteworthy given that Lhr is induced in mycobacteria exposed to the inter-strand crosslinker mitomycin C.


Assuntos
DNA Helicases/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Mycobacterium smegmatis/enzimologia , DNA Bacteriano/química , Domínios Proteicos , RNA Bacteriano/química
7.
PLoS One ; 16(1): e0245745, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33471823

RESUMO

Reduction of nitrate to nitrite in bacteria is an essential step in the nitrogen cycle, catalysed by a variety of nitrate reductase (NR) enzymes. The soil dweller, Mycobacterium smegmatis is able to assimilate nitrate and herein we set out to confirm the genetic basis for this by probing NR activity in mutants defective for putative nitrate reductase (NR) encoding genes. In addition to the annotated narB and narGHJI, bioinformatics identified three other putative NR-encoding genes: MSMEG_4206, MSMEG_2237 and MSMEG_6816. To assess the relative contribution of each, the corresponding gene loci were deleted using two-step allelic replacement, individually and in combination. The resulting strains were tested for their ability to assimilate nitrate and reduce nitrate under aerobic and anaerobic conditions, using nitrate assimilation and modified Griess assays. We demonstrated that narB, narGHJI, MSMEG_2237 and MSMEG_6816 were individually dispensable for nitrate assimilation and for nitrate reductase activity under aerobic and anaerobic conditions. Only deletion of MSMEG_4206 resulted in significant reduction in nitrate assimilation under aerobic conditions. These data confirm that in M. smegmatis, narB, narGHJI, MSMEG_2237 and MSMEG_6816 are not required for nitrate reduction as MSMEG_4206 serves as the sole assimilatory NR.


Assuntos
Proteínas de Bactérias/genética , Mycobacterium smegmatis/enzimologia , Nitrato Redutase/genética , Proteínas de Bactérias/metabolismo , Deleção de Genes , Mycobacterium smegmatis/genética , Nitrato Redutase/metabolismo , Nitratos/metabolismo
8.
Nat Commun ; 12(1): 143, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33420031

RESUMO

Coenzyme A (CoA) is a fundamental co-factor for all life, involved in numerous metabolic pathways and cellular processes, and its biosynthetic pathway has raised substantial interest as a drug target against multiple pathogens including Mycobacterium tuberculosis. The biosynthesis of CoA is performed in five steps, with the second and third steps being catalysed in the vast majority of prokaryotes, including M. tuberculosis, by a single bifunctional protein, CoaBC. Depletion of CoaBC was found to be bactericidal in M. tuberculosis. Here we report the first structure of a full-length CoaBC, from the model organism Mycobacterium smegmatis, describe how it is organised as a dodecamer and regulated by CoA thioesters. A high-throughput biochemical screen focusing on CoaB identified two inhibitors with different chemical scaffolds. Hit expansion led to the discovery of potent and selective inhibitors of M. tuberculosis CoaB, which we show to bind to a cryptic allosteric site within CoaB.


Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Carboxiliases/antagonistas & inibidores , Mycobacterium smegmatis/enzimologia , Mycobacterium tuberculosis/efeitos dos fármacos , Peptídeo Sintases/antagonistas & inibidores , Regulação Alostérica/efeitos dos fármacos , Sítio Alostérico/efeitos dos fármacos , Antituberculosos/uso terapêutico , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Carboxiliases/genética , Carboxiliases/metabolismo , Carboxiliases/ultraestrutura , Coenzima A/biossíntese , Cristalografia por Raios X , Ensaios Enzimáticos , Técnicas de Silenciamento de Genes , Ensaios de Triagem em Larga Escala , Humanos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Peptídeo Sintases/ultraestrutura , Tuberculose/tratamento farmacológico , Tuberculose/microbiologia
9.
Mol Biol Evol ; 38(3): 761-776, 2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32870983

RESUMO

The understanding of how proteins evolve to perform novel functions has long been sought by biologists. In this regard, two homologous bacterial enzymes, PafA and Dop, pose an insightful case study, as both rely on similar mechanistic properties, yet catalyze different reactions. PafA conjugates a small protein tag to target proteins, whereas Dop removes the tag by hydrolysis. Given that both enzymes present a similar fold and high sequence similarity, we sought to identify the differences in the amino acid sequence and folding responsible for each distinct activity. We tackled this question using analysis of sequence-function relationships, and identified a set of uniquely conserved residues in each enzyme. Reciprocal mutagenesis of the hydrolase, Dop, completely abolished the native activity, at the same time yielding a catalytically active ligase. Based on the available Dop and PafA crystal structures, this change of activity required a conformational change of a critical loop at the vicinity of the active site. We identified the conserved positions essential for stabilization of the alternative loop conformation, and tracked alternative mutational pathways that lead to a change in activity. Remarkably, all these pathways were combined in the evolution of PafA and Dop, despite their redundant effect on activity. Overall, we identified the residues and structural elements in PafA and Dop responsible for their activity differences. This analysis delineated, in molecular terms, the changes required for the emergence of a new catalytic function from a preexisting one.


Assuntos
Evolução Molecular , Hidrolases/genética , Ligases/genética , Mycobacterium smegmatis/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Escherichia coli , Hidrolases/química , Ligases/química , Conformação Proteica
10.
Nature ; 589(7840): 143-147, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33299175

RESUMO

Tuberculosis-the world's leading cause of death by infectious disease-is increasingly resistant to current first-line antibiotics1. The bacterium Mycobacterium tuberculosis (which causes tuberculosis) can survive low-energy conditions, allowing infections to remain dormant and decreasing their susceptibility to many antibiotics2. Bedaquiline was developed in 2005 from a lead compound identified in a phenotypic screen against Mycobacterium smegmatis3. This drug can sterilize even latent M. tuberculosis infections4 and has become a cornerstone of treatment for multidrug-resistant and extensively drug-resistant tuberculosis1,5,6. Bedaquiline targets the mycobacterial ATP synthase3, which is an essential enzyme in the obligate aerobic Mycobacterium genus3,7, but how it binds the intact enzyme is unknown. Here we determined cryo-electron microscopy structures of M. smegmatis ATP synthase alone and in complex with bedaquiline. The drug-free structure suggests that hook-like extensions from the α-subunits prevent the enzyme from running in reverse, inhibiting ATP hydrolysis and preserving energy in hypoxic conditions. Bedaquiline binding induces large conformational changes in the ATP synthase, creating tight binding pockets at the interface of subunits a and c that explain the potency of this drug as an antibiotic for tuberculosis.


Assuntos
Complexos de ATP Sintetase/química , Antituberculosos/química , Microscopia Crioeletrônica , Diarilquinolinas/química , Mycobacterium smegmatis/enzimologia , Tuberculose/tratamento farmacológico , Tuberculose/microbiologia , Complexos de ATP Sintetase/antagonistas & inibidores , Complexos de ATP Sintetase/metabolismo , Trifosfato de Adenosina/metabolismo , Antituberculosos/metabolismo , Antituberculosos/farmacologia , Diarilquinolinas/metabolismo , Diarilquinolinas/farmacologia , Hidrólise/efeitos dos fármacos , Modelos Moleculares , Mycobacterium smegmatis/efeitos dos fármacos , Rotação
11.
Nat Commun ; 11(1): 6419, 2020 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-33339823

RESUMO

RNA synthesis is central to life, and RNA polymerase (RNAP) depends on accessory factors for recovery from stalled states and adaptation to environmental changes. Here, we investigated the mechanism by which a helicase-like factor HelD recycles RNAP. We report a cryo-EM structure of a complex between the Mycobacterium smegmatis RNAP and HelD. The crescent-shaped HelD simultaneously penetrates deep into two RNAP channels that are responsible for nucleic acids binding and substrate delivery to the active site, thereby locking RNAP in an inactive state. We show that HelD prevents non-specific interactions between RNAP and DNA and dissociates stalled transcription elongation complexes. The liberated RNAP can either stay dormant, sequestered by HelD, or upon HelD release, restart transcription. Our results provide insights into the architecture and regulation of the highly medically-relevant mycobacterial transcription machinery and define HelD as a clearing factor that releases RNAP from nonfunctional complexes with nucleic acids.


Assuntos
Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Mycobacterium smegmatis/enzimologia , Ácidos Nucleicos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , Domínio Catalítico , Microscopia Crioeletrônica , DNA Bacteriano/química , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/ultraestrutura , Modelos Moleculares , Ligação Proteica , Domínios Proteicos
12.
J Enzyme Inhib Med Chem ; 35(1): 1964-1989, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33164573

RESUMO

Although trehalose has recently gained interest because of its pharmaceutical potential, its clinical use is hampered due to its low bioavailability. Hence, hydrolysis-resistant trehalose analogues retaining biological activity could be of interest. In this study, 34 4- and 6-O-substituted trehalose derivatives were synthesised using an ether- or carbamate-type linkage. Their hydrolysis susceptibility and inhibitory properties were determined against two trehalases, i.e. porcine kidney and Mycobacterium smegmatis. With the exception of three weakly hydrolysable 6-O-alkyl derivatives, the compounds generally showed to be completely resistant. Moreover, a number of derivatives was shown to be an inhibitor of one or both of these trehalases. For the strongest inhibitors of porcine kidney trehalase IC50 values of around 10 mM could be determined, whereas several compounds displayed sub-mM IC50 against M. smegmatis trehalase. Dockings studies were performed to explain the observed influence of the substitution pattern on the inhibitory activity towards porcine kidney trehalase.


Assuntos
Inibidores Enzimáticos/síntese química , Trealase/antagonistas & inibidores , Trealose/síntese química , Alquilação , Animais , Carbamatos/química , Inibidores Enzimáticos/metabolismo , Éter/química , Hidrólise , Rim/enzimologia , Simulação de Acoplamento Molecular , Mycobacterium smegmatis/enzimologia , Ligação Proteica , Relação Estrutura-Atividade , Suínos , Trealose/metabolismo
13.
Int J Mol Sci ; 21(22)2020 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-33198276

RESUMO

Nitric oxide (NO) is a well-known active site ligand and inhibitor of respiratory terminal oxidases. Here, we investigated the interaction of NO with a purified chimeric bcc-aa3 supercomplex composed of Mycobacterium tuberculosis cytochrome bcc and Mycobacterium smegmatisaa3-type terminal oxidase. Strikingly, we found that the enzyme in turnover with O2 and reductants is resistant to inhibition by the ligand, being able to metabolize NO at 25 °C with an apparent turnover number as high as ≈303 mol NO (mol enzyme)-1 min-1 at 30 µM NO. The rate of NO consumption proved to be proportional to that of O2 consumption, with 2.65 ± 0.19 molecules of NO being consumed per O2 molecule by the mycobacterial bcc-aa3. The enzyme was found to metabolize the ligand even under anaerobic reducing conditions with a turnover number of 2.8 ± 0.5 mol NO (mol enzyme)-1 min-1 at 25 °C and 8.4 µM NO. These results suggest a protective role of mycobacterial bcc-aa3 supercomplexes against NO stress.


Assuntos
Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Óxido Nítrico/farmacologia , Proteínas de Bactérias/metabolismo , Catálise , Domínio Catalítico , Transporte de Elétrons , Radicais Livres , Ligantes , Mycobacterium smegmatis/enzimologia , Mycobacterium tuberculosis/enzimologia , Óxido Nítrico/química , Oxirredutases/metabolismo , Oxigênio , Ligação Proteica
14.
Proc Natl Acad Sci U S A ; 117(38): 23794-23801, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32900960

RESUMO

Biotin plays an essential role in growth of mycobacteria. Synthesis of the cofactor is essential for Mycobacterium tuberculosis to establish and maintain chronic infections in a murine model of tuberculosis. Although the late steps of mycobacterial biotin synthesis, assembly of the heterocyclic rings, are thought to follow the canonical pathway, the mechanism of synthesis of the pimelic acid moiety that contributes most of the biotin carbon atoms is unknown. We report that the Mycobacterium smegmatis gene annotated as encoding Tam, an O-methyltransferase that monomethylates and detoxifies trans-aconitate, instead encodes a protein having the activity of BioC, an O-methyltransferase that methylates the free carboxyl of malonyl-ACP. The M. smegmatis Tam functionally replaced Escherichia coli BioC both in vivo and in vitro. Moreover, deletion of the M. smegmatis tam gene resulted in biotin auxotrophy, and addition of biotin to M. smegmatis cultures repressed tam gene transcription. Although its pathogenicity precluded in vivo studies, the M. tuberculosis Tam also replaced E. coli BioC both in vivo and in vitro and complemented biotin-independent growth of the M. smegmatis tam deletion mutant strain. Based on these data, we propose that the highly conserved mycobacterial tam genes be renamed bioC M. tuberculosis BioC presents a target for antituberculosis drugs which thus far have been directed at late reactions in the pathway with some success.


Assuntos
Biotina/biossíntese , Mycobacterium smegmatis , Mycobacterium tuberculosis , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli , Redes e Vias Metabólicas , Mycobacterium smegmatis/enzimologia , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Proteína O-Metiltransferase
15.
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
16.
Int J Mol Sci ; 21(15)2020 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-32751237

RESUMO

Several alkylating agents that either occur in the environment or are self-produced can cause DNA-damaging injuries in bacterial cells. Therefore, all microorganisms have developed repair systems that are able to counteract DNA alkylation damage. The adaptive response to alkylation stress in Escherichia coli consists of the Ada operon, which has been widely described; however, the homologous system in Mycobacterium tuberculosis (MTB) has been shown to have a different genetic organization but it is still largely unknown. In order to describe the defense system of MTB, we first investigated the proteins involved in the repair mechanism in the homologous non-pathogenic mycobacterium M. smegmatis. Ogt, Ada-AlkA and FadE8 proteins were recombinantly produced, purified and characterized. The biological role of Ogt was examined using proteomic experiments to identify its protein partners in vivo under stress conditions. Our results suggested the formation of a functional complex between Ogt and Ada-AlkA, which was confirmed both in silico by docking calculations and by gel filtration chromatography. We propose that this stable association allows the complex to fulfill the biological roles exerted by Ada in the homologous E. coli system. Finally, FadE8 was demonstrated to be structurally and functionally related to its E. coli homologous, AidB.


Assuntos
Acil-CoA Desidrogenase/química , Proteínas de Bactérias/química , Reparo do DNA , DNA Bacteriano/genética , Metiltransferases/química , Mycobacterium smegmatis/genética , Acil-CoA Desidrogenase/genética , Acil-CoA Desidrogenase/metabolismo , Alquilantes/farmacologia , Alquilação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cromossomos Bacterianos/química , Clonagem Molecular , Dano ao DNA , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo , Simulação de Acoplamento Molecular , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/enzimologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteômica/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
17.
Biomolecules ; 10(8)2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32751900

RESUMO

NADPH-dependent imine reductases (IREDs) are enzymes capable of enantioselectively reducing imines to chiral secondary amines, which represent important building blocks in the chemical and pharmaceutical industry. Since their discovery in 2011, many previously unknown IREDs have been identified, biochemically and structurally characterized and categorized into families. However, the catalytic mechanism and guiding principles for substrate specificity and stereoselectivity remain disputed. Herein, we describe the crystal structure of S-IRED-Ms from Mycobacterium smegmatis together with its cofactor NADPH. S-IRED-Ms belongs to the S-enantioselective superfamily 3 (SFam3) and is the first IRED from SFam3 to be structurally described. The data presented provide further evidence for the overall high degree of structural conservation between different IREDs of various superfamilies. We discuss the role of Asp170 in catalysis and the importance of hydrophobic amino acids in the active site for stereospecificity. Moreover, a separate entrance to the active site, potentially functioning according to a gatekeeping mechanism regulating access and, therefore, substrate specificity is described.


Assuntos
Proteínas de Bactérias/química , Iminas/metabolismo , Mycobacterium smegmatis/enzimologia , Oxirredutases/química , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Iminas/química , NADP/química , NADP/metabolismo , Oxirredutases/metabolismo , Ligação Proteica , Estereoisomerismo , Especificidade por Substrato
18.
Nat Commun ; 11(1): 3396, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32636380

RESUMO

Arabinosyltransferase B (EmbB) belongs to a family of membrane-bound glycosyltransferases that build the lipidated polysaccharides of the mycobacterial cell envelope, and are targets of anti-tuberculosis drug ethambutol. We present the 3.3 Å resolution single-particle cryo-electron microscopy structure of Mycobacterium smegmatis EmbB, providing insights on substrate binding and reaction mechanism. Mutations that confer ethambutol resistance map mostly around the putative active site, suggesting this to be the location of drug binding.


Assuntos
Mycobacterium smegmatis/enzimologia , Pentosiltransferases/química , Pentosiltransferases/ultraestrutura , Antituberculosos/farmacologia , Domínio Catalítico , Microscopia Crioeletrônica , Farmacorresistência Bacteriana , Etambutol/farmacologia , Lipídeos/química , Mutação , Mycobacterium tuberculosis/enzimologia , Polissacarídeos/química , Ligação Proteica
19.
J Biol Chem ; 295(29): 9868-9878, 2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32434931

RESUMO

Fold-switch pathways remodel the secondary structure topology of proteins in response to the cellular environment. It is a major challenge to understand the dynamics of these folding processes. Here, we conducted an in-depth analysis of the α-helix-to-ß-strand and ß-strand-to-α-helix transitions and domain motions displayed by the essential mannosyltransferase PimA from mycobacteria. Using 19F NMR, we identified four functionally relevant states of PimA that coexist in dynamic equilibria on millisecond-to-second timescales in solution. We discovered that fold-switching is a slow process, on the order of seconds, whereas domain motions occur simultaneously but are substantially faster, on the order of milliseconds. Strikingly, the addition of substrate accelerated the fold-switching dynamics of PimA. We propose a model in which the fold-switching dynamics constitute a mechanism for PimA activation.


Assuntos
Proteínas de Bactérias/química , Manosiltransferases/química , Simulação de Dinâmica Molecular , Mycobacterium smegmatis/enzimologia , Dobramento de Proteína , Ressonância Magnética Nuclear Biomolecular
20.
Biochem Biophys Res Commun ; 527(1): 37-41, 2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32446388

RESUMO

The tryptophan biosynthesis pathway, which does not exist in mammals, is highly conserved in Mycobacterium. Anthranilate synthase (AS) catalyzes the initial reactions in the tryptophan biosynthesis pathway in many microorganisms, catalyzing the conversion of glutamine and chorismate to form pyruvate and anthranilate. Here, the crystal structure of anthranilate synthase component I (AS I) from Mycolicibacterium smegmatis (MsTrpE) has been determined to 1.7 Å resolution. MsTrpE crystallizes in the space group P1 with two monomers in the asymmetric unit, which is consistent with the oligomeric state in solution as confirmed by analytical ultracentrifugation. Inspection of the active site shows that it is in the active form with a bound Mg2+ ion and a ligand that is modelled as benzoate. The position of benzoate mimics the position of the anthranilate product in the active site. The structure of MsTrpE will provide a starting point for the investigation of latent biotechnology and pharmaceutical applications of anthranilate synthase component I.


Assuntos
Antranilato Sintase/química , Proteínas de Bactérias/química , Mycobacterium smegmatis/enzimologia , Cristalografia por Raios X , Humanos , Modelos Moleculares , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium smegmatis/química , Conformação Proteica , Subunidades Proteicas/química
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