RESUMO
In many gram-positive Actinobacteria, including Actinomyces oris and Corynebacterium matruchotii, the conserved thiol-disulfide oxidoreductase MdbA that catalyzes oxidative folding of exported proteins is essential for bacterial viability by an unidentified mechanism. Intriguingly, in Corynebacterium diphtheriae, the deletion of mdbA blocks cell growth only at 37 °C but not at 30 °C, suggesting the presence of alternative oxidoreductase enzyme(s). By isolating spontaneous thermotolerant revertants of the mdbA mutant at 37 °C, we obtained genetic suppressors, all mapped to a single T-to-G mutation within the promoter region of tsdA, causing its elevated expression. Strikingly, increased expression of tsdA-via suppressor mutations or a constitutive promoter-rescues the pilus assembly and toxin production defects of this mutant, hence compensating for the loss of mdbA. Structural, genetic, and biochemical analyses demonstrated TsdA is a membrane-tethered thiol-disulfide oxidoreductase with a conserved CxxC motif that can substitute for MdbA in mediating oxidative folding of pilin and toxin substrates. Together with our observation that tsdA expression is upregulated at nonpermissive temperature (40 °C) in wild-type cells, we posit that TsdA has evolved as a compensatory thiol-disulfide oxidoreductase that safeguards oxidative protein folding in C. diphtheriae against thermal stress.
Assuntos
Proteínas de Bactérias , Corynebacterium diphtheriae , Proteína Dissulfeto Redutase (Glutationa) , Dobramento de Proteína , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Corynebacterium diphtheriae/enzimologia , Corynebacterium diphtheriae/genética , Estresse Oxidativo , Proteína Dissulfeto Redutase (Glutationa)/genética , Proteína Dissulfeto Redutase (Glutationa)/metabolismoRESUMO
The biphasic assembly of Gram-positive pili begins with the covalent polymerization of distinct pilins catalyzed by a pilus-specific sortase, followed by the cell wall anchoring of the resulting polymers mediated by the housekeeping sortase. In Actinomyces oris, the pilus-specific sortase SrtC2 not only polymerizes FimA pilins to assemble type 2 fimbriae with CafA at the tip, but it can also act as the anchoring sortase, linking both FimA polymers and SrtC1-catalyzed FimP polymers (type 1 fimbriae) to peptidoglycan when the housekeeping sortase SrtA is inactive. To date, the structure-function determinants governing the unique substrate specificity and dual enzymatic activity of SrtC2 have not been illuminated. Here, we present the crystal structure of SrtC2 solved to 2.10-Å resolution. SrtC2 harbors a canonical sortase fold and a lid typical for class C sortases and additional features specific to SrtC2. Structural, biochemical, and mutational analyses of SrtC2 reveal that the extended lid of SrtC2 modulates its dual activity. Specifically, we demonstrate that the polymerizing activity of SrtC2 is still maintained by alanine-substitution, partial deletion, and replacement of the SrtC2 lid with the SrtC1 lid. Strikingly, pilus incorporation of CafA is significantly reduced by these mutations, leading to compromised polymicrobial interactions mediated by CafA. In a srtA mutant, the partial deletion of the SrtC2 lid reduces surface anchoring of FimP polymers, and the lid-swapping mutation enhances this process, while both mutations diminish surface anchoring of FimA pili. Evidently, the extended lid of SrtC2 enables the enzyme the cell wall-anchoring activity in a substrate-selective fashion.
Assuntos
Aminoaciltransferases , Proteínas de Bactérias , Cisteína Endopeptidases , Proteínas de Fímbrias , Fímbrias Bacterianas , Cisteína Endopeptidases/metabolismo , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Aminoaciltransferases/metabolismo , Aminoaciltransferases/genética , Aminoaciltransferases/química , Fímbrias Bacterianas/metabolismo , Fímbrias Bacterianas/genética , Proteínas de Fímbrias/metabolismo , Proteínas de Fímbrias/química , Proteínas de Fímbrias/genética , Cristalografia por Raios X , Actinomyces/metabolismo , Actinomyces/enzimologia , Especificidade por Substrato , Modelos MolecularesRESUMO
Assembly of pili on the gram-positive bacterial cell wall involves 2 conserved transpeptidase enzymes named sortases: One for polymerization of pilin subunits and another for anchoring pili to peptidoglycan. How this machine controls pilus length and whether pilus length is critical for cell-to-cell interactions remain unknown. We report here in Actinomyces oris, a key colonizer in the development of oral biofilms, that genetic disruption of its housekeeping sortase SrtA generates exceedingly long pili, catalyzed by its pilus-specific sortase SrtC2 that possesses both pilus polymerization and cell wall anchoring functions. Remarkably, the srtA-deficient mutant fails to mediate interspecies interactions, or coaggregation, even though the coaggregation factor CafA is present at the pilus tip. Increasing ectopic expression of srtA in the mutant progressively shortens pilus length and restores coaggregation accordingly, while elevated levels of shaft pilins and SrtC2 produce long pili and block coaggregation by SrtA+ bacteria. With structural studies, we uncovered 2 key structural elements in SrtA that partake in recognition of pilin substrates and regulate pilus length by inducing the capture and transfer of pilus polymers to the cell wall. Evidently, coaggregation requires proper positioning of the tip adhesin CafA via modulation of pilus length by the housekeeping sortase SrtA.
Assuntos
Actinomyces , Adesinas Bacterianas , Aminoaciltransferases , Proteínas de Bactérias , Cisteína Endopeptidases , Fímbrias Bacterianas , Actinomyces/química , Actinomyces/genética , Actinomyces/metabolismo , Adesinas Bacterianas/química , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Aminoaciltransferases/química , Aminoaciltransferases/genética , Aminoaciltransferases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Fímbrias Bacterianas/química , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/metabolismoRESUMO
Covalently cross-linked pilus polymers displayed on the cell surface of Gram-positive bacteria are assembled by class C sortase enzymes. These pilus-specific transpeptidases located on the bacterial membrane catalyze a two-step protein ligation reaction, first cleaving the LPXTG motif of one pilin protomer to form an acyl-enzyme intermediate and then joining the terminal Thr to the nucleophilic Lys residue residing within the pilin motif of another pilin protomer. To date, the determinants of class C enzymes that uniquely enable them to construct pili remain unknown. Here, informed by high-resolution crystal structures of corynebacterial pilus-specific sortase (SrtA) and utilizing a structural variant of the enzyme (SrtA2M), whose catalytic pocket has been unmasked by activating mutations, we successfully reconstituted in vitro polymerization of the cognate major pilin (SpaA). Mass spectrometry, electron microscopy, and biochemical experiments authenticated that SrtA2M synthesizes pilus fibers with correct Lys-Thr isopeptide bonds linking individual pilins via a thioacyl intermediate. Structural modeling of the SpaA-SrtA-SpaA polymerization intermediate depicts SrtA2M sandwiched between the N- and C-terminal domains of SpaA harboring the reactive pilin and LPXTG motifs, respectively. Remarkably, the model uncovered a conserved TP(Y/L)XIN(S/T)H signature sequence following the catalytic Cys, in which the alanine substitutions abrogated cross-linking activity but not cleavage of LPXTG. These insights and our evidence that SrtA2M can terminate pilus polymerization by joining the terminal pilin SpaB to SpaA and catalyze ligation of isolated SpaA domains in vitro provide a facile and versatile platform for protein engineering and bio-conjugation that has major implications for biotechnology.
Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Corynebacterium/metabolismo , Cisteína Endopeptidases/metabolismo , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Catálise , Parede Celular/metabolismo , Cristalografia por Raios X/métodos , Peptidil Transferases/metabolismo , PolimerizaçãoRESUMO
The flavin transferase ApbE plays essential roles in bacterial physiology, covalently incorporating FMN cofactors into numerous respiratory enzymes that use the integrated cofactors as electron carriers. In this work we performed a detailed kinetic and structural characterization of Vibrio cholerae WT ApbE and mutants of the conserved residue His-257, to understand its role in substrate binding and in the catalytic mechanism of this family. Bi-substrate kinetic experiments revealed that ApbE follows a random Bi Bi sequential kinetic mechanism, in which a ternary complex is formed, indicating that both substrates must be bound to the enzyme for the reaction to proceed. Steady-state kinetic analyses show that the turnover rates of His-257 mutants are significantly smaller than those of WT ApbE, and have increased Km values for both substrates, indicating that the His-257 residue plays important roles in catalysis and in enzyme-substrate complex formation. Analyses of the pH dependence of ApbE activity indicate that the pKa of the catalytic residue (pKES1) increases by 2 pH units in the His-257 mutants, suggesting that this residue plays a role in substrate deprotonation. The crystal structures of WT ApbE and an H257G mutant were determined at 1.61 and 1.92 Å resolutions, revealing that His-257 is located in the catalytic site and that the substitution does not produce major conformational changes. We propose a reaction mechanism in which His-257 acts as a general base that deprotonates the acceptor residue, which subsequently performs a nucleophilic attack on FAD for flavin transfer.
Assuntos
Flavinas/metabolismo , Transferases/metabolismo , Vibrio cholerae/metabolismo , Proteínas de Bactérias/metabolismo , Catálise , Domínio Catalítico , Sequência Conservada , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Flavinas/genética , Histidina/metabolismo , Cinética , Oxirredução , Especificidade por Substrato/genética , Transferases/genética , Vibrio cholerae/genéticaRESUMO
Acyl-coenzyme A (CoA) ligases catalyze the activation of carboxylic acids via a two-step reaction of adenylation followed by thioesterification. Here, we report the discovery of a non-adenylating acyl-CoA ligase PtmA2 and the functional separation of an acyl-CoA ligase reaction. Both PtmA1 and PtmA2, two acyl-CoA ligases from the biosynthetic pathway of platensimycin and platencin, are necessary for the two steps of CoA activation. Gene inactivation of ptmA1 and ptmA2 resulted in the accumulation of free acid and adenylate intermediates, respectively. Enzymatic and structural characterization of PtmA2 confirmed its ability to only catalyze thioesterification. Structural characterization of PtmA2 revealed it binds both free acid and adenylate substrates and undergoes the established mechanism of domain alternation. Finally, site-directed mutagenesis restored both the adenylation and complete CoA activation reactions. This study challenges the currently accepted paradigm of adenylating enzymes and inspires future investigations on functionally separated acyl-CoA ligases and their ramifications in biology.
Assuntos
Coenzima A Ligases/metabolismo , Biocatálise , Ácidos Carboxílicos/química , Ácidos Carboxílicos/metabolismo , Coenzima A Ligases/química , Coenzima A Ligases/isolamento & purificação , Ésteres/química , Ésteres/metabolismo , Modelos Moleculares , Estrutura Molecular , Compostos de Sulfidrila/química , Compostos de Sulfidrila/metabolismoRESUMO
The actinobacterium Corynebacterium matruchotii has been implicated in nucleation of oral microbial consortia leading to biofilm formation. Due to the lack of genetic tools, little is known about basic cellular processes, including protein secretion and folding, in this organism. We report here a survey of the C. matruchotii genome, which encodes a large number of exported proteins containing paired cysteine residues, and identified an oxidoreductase that is highly homologous to the Corynebacterium diphtheriae thiol-disulfide oxidoreductase MdbA (MdbACd). Crystallization studies uncovered that the 1.2-Å resolution structure of C. matruchotii MdbA (MdbACm) possesses two conserved features found in actinobacterial MdbA enzymes, a thioredoxin-like fold and an extended α-helical domain. By reconstituting the disulfide bond-forming machine in vitro, we demonstrated that MdbACm catalyzes disulfide bond formation within the actinobacterial pilin FimA. A new gene deletion method supported that mdbA is essential in C. matruchotii Remarkably, heterologous expression of MdbACm in the C. diphtheriae ΔmdbA mutant rescued its known defects in cell growth and morphology, toxin production, and pilus assembly, and this thiol-disulfide oxidoreductase activity required the catalytic motif CXXC. Altogether, the results suggest that MdbACm is a major thiol-disulfide oxidoreductase, which likely mediates posttranslocational protein folding in C. matruchotii by a mechanism that is conserved in ActinobacteriaIMPORTANCE The actinobacterium Corynebacterium matruchotii has been implicated in the development of oral biofilms or dental plaque; however, little is known about the basic cellular processes in this organism. We report here a high-resolution structure of a C. matruchotii oxidoreductase that is highly homologous to the Corynebacterium diphtheriae thiol-disulfide oxidoreductase MdbA. By biochemical analysis, we demonstrated that C. matruchotii MdbA catalyzes disulfide bond formation in vitro Furthermore, a new gene deletion method revealed that deletion of mdbA is lethal in C. matruchotii Remarkably, C. matruchotii MdbA can replace C. diphtheriae MdbA to maintain normal cell growth and morphology, toxin production, and pilus assembly. Overall, our studies support the hypothesis that C. matruchotii utilizes MdbA as a major oxidoreductase to catalyze oxidative protein folding.
Assuntos
Proteínas de Bactérias/química , Corynebacterium/enzimologia , Corynebacterium/genética , Proteína Dissulfeto Redutase (Glutationa)/química , Proteínas de Bactérias/genética , Biofilmes , Catálise , Corynebacterium diphtheriae/enzimologia , Corynebacterium diphtheriae/genética , Dissulfetos/química , Proteínas de Fímbrias/química , Proteínas de Fímbrias/genética , Deleção de Genes , Genoma Bacteriano , Modelos Moleculares , Oxirredução , Oxirredutases/química , Oxirredutases/genética , Proteína Dissulfeto Redutase (Glutationa)/genéticaRESUMO
The X-ray crystal structure of the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase from Haemophilus influenzae (HiDapE) bound by the products of hydrolysis, succinic acid and l,l-DAP, was determined at 1.95 Å. Surprisingly, the structure bound to the products revealed that HiDapE undergoes a significant conformational change in which the catalytic domain rotates â¼50° and shifts â¼10.1 Å (as measured at the position of the Zn atoms) relative to the dimerization domain. This heretofore unobserved closed conformation revealed significant movements within the catalytic domain compared to that of wild-type HiDapE, which results in effectively closing off access to the dinuclear Zn(II) active site with the succinate carboxylate moiety bridging the dinculear Zn(II) cluster in a µ-1,3 fashion forming a bis(µ-carboxylato)dizinc(II) core with a Zn-Zn distance of 3.8 Å. Surprisingly, His194.B, which is located on the dimerization domain of the opposing chain â¼10.1 Å from the dinuclear Zn(II) active site, forms a hydrogen bond (2.9 Å) with the oxygen atom of succinic acid bound to Zn2, forming an oxyanion hole. As the closed structure forms upon substrate binding, the movement of His194.B by more than â¼10 Å is critical, based on site-directed mutagenesis data, for activation of the scissile carbonyl carbon of the substrate for nucleophilic attack by a hydroxide nucleophile. Employing the HiDapE product-bound structure as the starting point, a reverse engineering approach called product-based transition-state modeling provided structural models for each major catalytic step. These data provide insight into the catalytic reaction mechanism and also the future design of new, potent inhibitors of DapE enzymes.
Assuntos
Amidoidrolases/química , Proteínas de Bactérias/química , Haemophilus influenzae/enzimologia , Amidoidrolases/genética , Amidoidrolases/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Catálise , Domínio Catalítico , Cristalografia por Raios X , Ácido Diaminopimélico/metabolismo , Dimerização , Haemophilus influenzae/genética , Ligação de Hidrogênio , Modelos Moleculares , Mutagênese Sítio-Dirigida , Neisseria meningitidis/enzimologia , Neisseria meningitidis/genética , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/química , Rotação , Especificidade por Substrato , Ácido Succínico/metabolismo , Zinco/químicaRESUMO
Proteins that are site-specifically modified with peptides and chemicals can be used as novel therapeutics, imaging tools, diagnostic reagents and materials. However, there are few enzyme-catalyzed methods currently available to selectively conjugate peptides to internal sites within proteins. Here we show that a pilus-specific sortase enzyme from Corynebacterium diphtheriae (CdSrtA) can be used to attach a peptide to a protein via a specific lysine-isopeptide bond. Using rational mutagenesis we created CdSrtA3M, a highly activated cysteine transpeptidase that catalyzes in vitro isopeptide bond formation. CdSrtA3M mediates bioconjugation to a specific lysine residue within a fused domain derived from the corynebacterial SpaA protein. Peptide modification yields greater than >95% can be achieved. We demonstrate that CdSrtA3M can be used in concert with the Staphylococcus aureus SrtA enzyme, enabling dual, orthogonal protein labeling via lysine-isopeptide and backbone-peptide bonds.
Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Corynebacterium diphtheriae/enzimologia , Cisteína Endopeptidases/metabolismo , Corantes Fluorescentes/metabolismo , Lisina/metabolismo , Peptídeos/metabolismo , Proteínas de Bactérias/química , Corynebacterium diphtheriae/metabolismo , Proteínas de Fímbrias/metabolismo , Corantes Fluorescentes/química , Lisina/química , Modelos Moleculares , Peptídeos/química , Polimerização , Coloração e Rotulagem , Staphylococcus aureus/enzimologiaRESUMO
Acyltransferase (AT)-less type I polyketide synthases (PKSs) break the type I PKS paradigm. They lack the integrated AT domains within their modules and instead use a discrete AT that acts in trans, whereas a type I PKS module minimally contains AT, acyl carrier protein (ACP), and ketosynthase (KS) domains. Structures of canonical type I PKS KS-AT didomains reveal structured linkers that connect the two domains. AT-less type I PKS KSs have remnants of these linkers, which have been hypothesized to be AT docking domains. Natural products produced by AT-less type I PKSs are very complex because of an increased representation of unique modifying domains. AT-less type I PKS KSs possess substrate specificity and fall into phylogenetic clades that correlate with their substrates, whereas canonical type I PKS KSs are monophyletic. We have solved crystal structures of seven AT-less type I PKS KS domains that represent various sequence clusters, revealing insight into the large structural and subtle amino acid residue differences that lead to unique active site topologies and substrate specificities. One set of structures represents a larger group of KS domains from both canonical and AT-less type I PKSs that accept amino acid-containing substrates. One structure has a partial AT-domain, revealing the structural consequences of a type I PKS KS evolving into an AT-less type I PKS KS. These structures highlight the structural diversity within the AT-less type I PKS KS family, and most important, provide a unique opportunity to study the molecular evolution of substrate specificity within the type I PKSs.
Assuntos
Evolução Molecular , Policetídeo Sintases/química , Cristalografia por Raios X , Policetídeo Sintases/genética , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
Pathogens deliver complex arsenals of translocated effector proteins to host cells during infection, but the extent to which these proteins are regulated once inside the eukaryotic cell remains poorly defined. Among all bacterial pathogens, Legionella pneumophila maintains the largest known set of translocated substrates, delivering over 300 proteins to the host cell via its Type IVB, Icm/Dot translocation system. Backed by a few notable examples of effector-effector regulation in L. pneumophila, we sought to define the extent of this phenomenon through a systematic analysis of effector-effector functional interaction. We used Saccharomyces cerevisiae, an established proxy for the eukaryotic host, to query > 108,000 pairwise genetic interactions between two compatible expression libraries of ~330 L. pneumophila-translocated substrates. While capturing all known examples of effector-effector suppression, we identify fourteen novel translocated substrates that suppress the activity of other bacterial effectors and one pair with synergistic activities. In at least nine instances, this regulation is direct-a hallmark of an emerging class of proteins called metaeffectors, or "effectors of effectors". Through detailed structural and functional analysis, we show that metaeffector activity derives from a diverse range of mechanisms, shapes evolution, and can be used to reveal important aspects of each cognate effector's function. Metaeffectors, along with other, indirect, forms of effector-effector modulation, may be a common feature of many intracellular pathogens-with unrealized potential to inform our understanding of how pathogens regulate their interactions with the host cell.
Assuntos
Proteínas de Bactérias/metabolismo , Legionella pneumophila/patogenicidade , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Interações Hospedeiro-Patógeno , Legionella pneumophila/metabolismo , Modelos Biológicos , Mapas de Interação de Proteínas , Biologia de Sistemas/métodosRESUMO
Multidrug transcription regulator AcrR from Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 belongs to the tetracycline repressor family, one of the largest groups of bacterial transcription factors. The crystal structure of dimeric AcrR was determined and refined to 1.56Å resolution. The tertiary and quaternary structures of AcrR are similar to those of its homologs. The multidrug binding site was identified based on structural alignment with homologous proteins and has a di(hydroxyethyl)ether molecule bound. Residues from helices α4 and α7 shape the entry into this binding site. The structure of AcrR reveals that the extended helical conformation of helix α4 is stabilized by the hydrogen bond between Glu67 (helix α4) and Gln130 (helix α7). Based on the structural comparison with the closest homolog structure, the Escherichia coli AcrR, we propose that this hydrogen bond is responsible for control of the loop-to-helix transition within helix α4. This local conformational switch of helix α4 may be a key step in accessing the multidrug binding site and securing ligands at the binding site. Solution small-molecule binding studies suggest that AcrR binds ligands with their core chemical structure resembling the tetracyclic ring of cholesterol.
Assuntos
Proteínas de Bactérias/química , Domínios Proteicos , Estrutura Secundária de Proteína , Proteínas Repressoras/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Ligação de Hidrogênio , Ligantes , Modelos Moleculares , Ligação Proteica , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Homologia de Sequência do Ácido NucleicoRESUMO
Export of cell surface pilins in Gram-positive bacteria likely occurs by the translocation of unfolded precursor polypeptides; however, how the unfolded pilins gain their native conformation is presently unknown. Here, we present physiological studies to demonstrate that the FimA pilin of Actinomyces oris contains two disulfide bonds. Alanine substitution of cysteine residues forming the C-terminal disulfide bridge abrogates pilus assembly, in turn eliminating biofilm formation and polymicrobial interaction. Transposon mutagenesis of A. oris yielded a mutant defective in adherence to Streptococcus oralis, and revealed the essential role of a vitamin K epoxide reductase (VKOR) gene in pilus assembly. Targeted deletion of vkor results in the same defects, which are rescued by ectopic expression of VKOR, but not a mutant containing an alanine substitution in its conserved CXXC motif. Depletion of mdbA, which encodes a membrane-bound thiol-disulfide oxidoreductase, abrogates pilus assembly and alters cell morphology. Remarkably, overexpression of MdbA or a counterpart from Corynebacterium diphtheriae, rescues the Δvkor mutant. By alkylation assays, we demonstrate that VKOR is required for MdbA reoxidation. Furthermore, crystallographic studies reveal that A. oris MdbA harbors a thioredoxin-like fold with the conserved CXXC active site. Consistently, each MdbA enzyme catalyzes proper disulfide bond formation within FimA in vitro that requires the catalytic CXXC motif. Because the majority of signal peptide-containing proteins encoded by A. oris possess multiple Cys residues, we propose that MdbA and VKOR constitute a major folding machine for the secretome of this organism. This oxidative protein folding pathway may be a common feature in Actinobacteria.
Assuntos
Actinomyces/fisiologia , Proteínas de Bactérias/metabolismo , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Proteína Dissulfeto Redutase (Glutationa)/metabolismo , Vitamina K Epóxido Redutases/metabolismo , Actinomyces/química , Actinomyces/citologia , Actinomyces/genética , Actinomicose/microbiologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biofilmes/efeitos dos fármacos , Cristalografia por Raios X , Dissulfetos/química , Dissulfetos/metabolismo , Proteínas de Fímbrias/química , Fímbrias Bacterianas/química , Deleção de Genes , Humanos , Interações Microbianas , Modelos Moleculares , Conformação Proteica , Proteína Dissulfeto Redutase (Glutationa)/química , Proteína Dissulfeto Redutase (Glutationa)/genética , Dobramento de Proteína , Vitamina K Epóxido Redutases/química , Vitamina K Epóxido Redutases/genéticaRESUMO
The steadily rising frequency of emerging diseases and antibiotic resistance creates an urgent need for new drugs and targets. Inosine 5'-monophosphate dehydrogenase (IMP dehydrogenase or IMPDH) is a promising target for the development of new antimicrobial agents. IMPDH catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD(+), which is the pivotal step in the biosynthesis of guanine nucleotides. Potent inhibitors of bacterial IMPDHs have been identified that bind in a structurally distinct pocket that is absent in eukaryotic IMPDHs. The physiological role of this pocket was not understood. Here, we report the structures of complexes with different classes of inhibitors of Bacillus anthracis, Campylobacter jejuni, and Clostridium perfringens IMPDHs. These structures in combination with inhibition studies provide important insights into the interactions that modulate selectivity and potency. We also present two structures of the Vibrio cholerae IMPDH in complex with IMP/NAD(+) and XMP/NAD(+). In both structures, the cofactor assumes a dramatically different conformation than reported previously for eukaryotic IMPDHs and other dehydrogenases, with the major change observed for the position of the NAD(+) adenosine moiety. More importantly, this new NAD(+)-binding site involves the same pocket that is utilized by the inhibitors. Thus, the bacterial IMPDH-specific NAD(+)-binding mode helps to rationalize the conformation adopted by several classes of prokaryotic IMPDH inhibitors. These findings offer a potential strategy for further ligand optimization.
Assuntos
Anti-Infecciosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , IMP Desidrogenase/antagonistas & inibidores , Sequência de Aminoácidos , Anti-Infecciosos/química , Bacillus anthracis/efeitos dos fármacos , Bacillus anthracis/enzimologia , Bacillus anthracis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Campylobacter jejuni/efeitos dos fármacos , Campylobacter jejuni/enzimologia , Campylobacter jejuni/genética , Clostridium perfringens/efeitos dos fármacos , Clostridium perfringens/enzimologia , Clostridium perfringens/genética , Cristalografia por Raios X , Inibidores Enzimáticos/química , IMP Desidrogenase/química , IMP Desidrogenase/genética , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Mutação , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Homologia de Sequência de AminoácidosRESUMO
Terpenoids are the largest and most structurally diverse family of natural products found in nature, yet their presence in bacteria is underappreciated. The carbon skeletons of terpenoids are generated through carbocation-dependent cyclization cascades catalyzed by terpene synthases (TSs). Type I and type II TSs initiate cyclization via diphosphate ionization and protonation, respectively, and protein structures of both types are known. Most plant diterpene synthases (DTSs) possess three α-helical domains (αßγ), which are thought to have arisen from the fusion of discrete, ancestral bacterial type I TSs (α) and type II TSs (ßγ). Type II DTSs of bacterial origin, of which there are no structurally characterized members, are a missing piece in the structural evolution of TSs. Here, we report the first crystal structure of a type II DTS from bacteria. PtmT2 from Streptomyces platensis CB00739 was verified as an ent-copalyl diphosphate synthase involved in the biosynthesis of platensimycin and platencin. The crystal structure of PtmT2 was solved at a resolution of 1.80 Å, and docking studies suggest the catalytically active conformation of geranylgeranyl diphosphate (GGPP). Site-directed mutagenesis confirmed residues involved in binding the diphosphate moiety of GGPP and identified DxxxxE as a potential Mg(2+)-binding motif for type II DTSs of bacterial origin. Finally, both the shape and physicochemical properties of the active sites are responsible for determining specific catalytic outcomes of TSs. The structure of PtmT2 fundamentally advances the knowledge of bacterial TSs, their mechanisms, and their role in the evolution of TSs.
Assuntos
Alquil e Aril Transferases/química , Alquil e Aril Transferases/metabolismo , Organofosfatos/metabolismo , Streptomyces/enzimologia , Domínio Catalítico , Modelos MolecularesRESUMO
The Bacillus anthracis virulence regulator AtxA controls transcription of the anthrax toxin genes and capsule biosynthetic operon. AtxA activity is elevated during growth in media containing glucose and CO(2)/bicarbonate, and there is a positive correlation between the CO(2)/bicarbonate signal, AtxA activity and homomultimerization. AtxA activity is also affected by phosphorylation at specific histidines. We show that AtxA crystallizes as a dimer. Distinct folds associated with predicted DNA-binding domains (HTH1 and HTH2) and phosphoenolpyruvate: carbohydrate phosphotransferase system-regulated domains (PRD1 and PRD2) are apparent. We tested AtxA variants containing single and double phosphomimetic (HisâAsp) and phosphoablative (HisâAla) amino acid changes for activity in B. anthracis cultures and for protein-protein interactions in cell lysates. Reduced activity of AtxA H199A, lack of multimerization and activity of AtxAH379D variants, and predicted structural changes associated with phosphorylation support a model for control of AtxA function. We propose that (i) in the AtxA dimer, phosphorylation of H199 in PRD1 affects HTH2 positioning, influencing DNA-binding; and (ii) phosphorylation of H379 in PRD2 disrupts dimer formation. The AtxA structure is the first reported high-resolution full-length structure of a PRD-containing regulator, and can serve as a model for proteins of this family, especially those that link virulence to bacterial metabolism.
Assuntos
Bacillus anthracis/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Histidina/metabolismo , Multimerização Proteica , Transativadores/química , Transativadores/metabolismo , Bacillus anthracis/genética , Bacillus anthracis/crescimento & desenvolvimento , Bacillus anthracis/patogenicidade , Cápsulas Bacterianas/metabolismo , Proteínas de Bactérias/genética , Cristalização , Cristalografia por Raios X , Modelos Moleculares , Mutação , Óperon , Fosforilação , Dobramento de Proteína , Estrutura Terciária de Proteína , Transativadores/genética , Virulência/genéticaRESUMO
The Gram-positive pathogen Corynebacterium diphtheriae exports through the Sec apparatus many extracellular proteins that include the key virulence factors diphtheria toxin and the adhesive pili. How these proteins attain their native conformations after translocation as unfolded precursors remains elusive. The fact that the majority of these exported proteins contain multiple cysteine residues and that several membrane-bound oxidoreductases are encoded in the corynebacterial genome suggests the existence of an oxidative protein-folding pathway in this organism. Here we show that the shaft pilin SpaA harbors a disulfide bond in vivo and alanine substitution of these cysteines abrogates SpaA polymerization and leads to the secretion of degraded SpaA peptides. We then identified a thiol-disulfide oxidoreductase (MdbA), whose structure exhibits a conserved thioredoxin-like domain with a CPHC active site. Remarkably, deletion of mdbA results in a severe temperature-sensitive cell division phenotype. This mutant also fails to assemble pilus structures and is greatly defective in toxin production. Consistent with these defects, the ΔmdbA mutant is attenuated in a guinea pig model of diphtheritic toxemia. Given its diverse cellular functions in cell division, pilus assembly and toxin production, we propose that MdbA is a component of the general oxidative folding machine in C. diphtheriae.
Assuntos
Corynebacterium diphtheriae/enzimologia , Corynebacterium diphtheriae/patogenicidade , Proteínas de Fímbrias/química , Proteínas de Fímbrias/metabolismo , Proteína Dissulfeto Redutase (Glutationa)/isolamento & purificação , Proteína Dissulfeto Redutase (Glutationa)/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Corynebacterium diphtheriae/fisiologia , Difteria/microbiologia , Toxina Diftérica/biossíntese , Toxina Diftérica/sangue , Fímbrias Bacterianas/química , Fímbrias Bacterianas/metabolismo , Cobaias , Viabilidade Microbiana , Mutação , Fenótipo , Proteína Dissulfeto Redutase (Glutationa)/química , Proteína Dissulfeto Redutase (Glutationa)/genética , Dobramento de Proteína , Toxemia/microbiologia , Virulência/genéticaRESUMO
Bacteria are becoming increasingly resistant to antibiotics, therefore there is an urgent need for new classes of antibiotics to fight antibiotic resistance. Mammals do not express N É -acetyl-L-ornithine deacetylase (ArgE), an enzyme that is critical for bacterial survival and growth, thus ArgE represents a promising new antibiotic drug target, as inhibitors would not suffer from mechanism-based toxicity. A new ninhydrin-based assay was designed and validated that included the synthesis of the substrate analog N 5, N 5-di-methyl N α-acetyl-L-ornithine (kcat/Km = 7.32 ± 0.94 × 104 M-1s-1). This new assay enabled the screening of potential inhibitors that absorb in the UV region, and thus is superior to the established 214 nm assay. Using this new ninhydrin-based assay, captopril was confirmed as an ArgE inhibitor (IC50 = 58.7 µM; Ki = 37.1 ± 0.85 µM), and a number of phenylboronic acid derivatives were identified as inhibitors, including 4-(diethylamino)phenylboronic acid (IC50 = 50.1 µM). Selected inhibitors were also tested in a thermal shift assay with ArgE using SYPRO Orange dye against Escherichia coli ArgE to observe the stability of the enzyme in the presence of inhibitors (captopril Ki = 35.9 ± 5.1 µM). The active site structure of di-Zn EcArgE was confirmed using X-ray absorption spectroscopy, and we reported two X-ray crystal structures of E. coli ArgE. In summary, we describe the development of a new ninhydrin-based assay for ArgE, the identification of captopril and phenylboronic acids as ArgE inhibitors, thermal shift studies with ArgE + captopril, and the first two published crystal structures of ArgE (mono-Zn and di-Zn).
RESUMO
[This corrects the article DOI: 10.3389/fchem.2024.1415644.].
RESUMO
The COVID-19 pandemic is caused by SARS-CoV-2, an RNA virus with high transmissibility and mutation rate. Given the paucity of orally bioavailable antiviral drugs to combat SARS-CoV-2 infection, there is a critical need for additional antivirals with alternative mechanisms of action. Papain-like protease (PLpro) is one of the two SARS-CoV-2 encoded viral cysteine proteases essential for viral replication. PLpro cleaves at three sites of the viral polyproteins. In addition, PLpro antagonizes the host immune response upon viral infection by cleaving ISG15 and ubiquitin from host proteins. Therefore, PLpro is a validated antiviral drug target. In this study, we report the X-ray crystal structures of papain-like protease (PLpro) with two potent inhibitors, Jun9722 and Jun9843. Subsequently, we designed and synthesized several series of analogs to explore the structure-activity relationship, which led to the discovery of PLpro inhibitors with potent enzymatic inhibitory activity and antiviral activity against SARS-CoV-2. Together, the lead compounds are promising drug candidates for further development.