RESUMO
Gram-positive bacteria assemble a multilayered cell wall that provides tensile strength to the cell. The cell wall is composed of glycan strands cross-linked by nonribosomally synthesized peptide stems. Herein, we modify the peptide stems of the Gram-positive bacterium Bacillus subtilis with noncanonical electrophilic d-amino acids, which when in proximity to adjacent stem peptides form novel covalent 5,3-cross-links. Approximately 20% of canonical cell-wall cross-links can be replaced with synthetic cross-links. While a low level of synthetic cross-link formation does not affect B. subtilis growth and phenotype, at higher levels cell growth is perturbed and bacteria elongate. A comparison of the accumulation of synthetic cross-links over time in Gram-negative and Gram-positive bacteria highlights key differences between them. The ability to perturb cell-wall architecture with synthetic building blocks provides a novel approach to studying the adaptability, elasticity, and porosity of bacterial cell walls.
Assuntos
Parede Celular/química , Bacilos Gram-Positivos/química , Peptidoglicano/química , Aminoácidos/química , Aminoácidos/metabolismo , Bacillus subtilis/química , Bacillus subtilis/citologia , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/metabolismo , Parede Celular/metabolismo , Bactérias Gram-Negativas/química , Bactérias Gram-Negativas/citologia , Bactérias Gram-Negativas/metabolismo , Bacilos Gram-Positivos/citologia , Bacilos Gram-Positivos/crescimento & desenvolvimento , Bacilos Gram-Positivos/metabolismo , Peptidoglicano/metabolismo , Peptidil Transferases/genética , Peptidil Transferases/metabolismo , FenótipoRESUMO
ß-Lactam antibiotics inhibit cell-wall transpeptidases, preventing the peptidoglycan, the major constituent of the bacterial cell wall, from cross-linking. This causes accumulation of long non-cross-linked strands of peptidoglycan, which leads to bacterial death. Pseudomonas aeruginosa, a nefarious bacterial pathogen, attempts to repair this aberrantly formed peptidoglycan by the function of the lytic transglycosylase Slt. We document in this report that Slt turns over the peptidoglycan by both exolytic and endolytic reactions, which cause glycosidic bond scission from a terminus or in the middle of the peptidoglycan, respectively. These reactions were characterized with complex synthetic peptidoglycan fragments that ranged in size from tetrasaccharides to octasaccharides. The X-ray structure of the wild-type apo Slt revealed it to be a doughnut-shaped protein. In a series of six additional X-ray crystal structures, we provide insights with authentic substrates into how Slt is enabled for catalysis for both the endolytic and exolytic reactions. The substrate for the exolytic reaction binds Slt in a canonical arrangement and reveals how both the glycan chain and the peptide stems are recognized by the Slt. We document that the apo enzyme does not have a fully formed active site for the endolytic reaction. However, binding of the peptidoglycan at the existing subsites within the catalytic domain causes a conformational change in the protein that assembles the surface for binding of a more expansive peptidoglycan between the catalytic domain and an adjacent domain. The complexes of Slt with synthetic peptidoglycan substrates provide an unprecedented snapshot of the endolytic reaction.
Assuntos
Proteínas de Bactérias/química , Glicosídeo Hidrolases/química , Peptidoglicano/química , Pseudomonas aeruginosa/enzimologia , Cristalografia por Raios X , Domínios Proteicos , Relação Estrutura-AtividadeRESUMO
The cell wall is an elaborate framework of peptidoglycan that serves to protect the bacterium against osmotic challenge. This exoskeleton is composed of repeating saccharides covalently cross-linked by peptide stems. The general structure of the cell wall is widely conserved across diverse Gram-negative bacteria. To begin to explore the biological consequence of introducing non-canonical cross-links into the cell wall of Escherichia coli, we generated a bacterium where up to 31% of the cell-wall cross-links are formed by a non-enzymatic reaction between a sulfonyl fluoride and an amino group. Bacteria with these non-canonical cell-wall cross-links achieve a high optical density in culture, divide and elongate successfully, and display no loss of outer membrane integrity. This work represents a first step in the design of bacteria with non-canonical "synthetic" cell walls.
Assuntos
Engenharia Celular/métodos , Parede Celular/metabolismo , Escherichia coli/metabolismo , Peptidoglicano/química , Peptidoglicano/metabolismo , Fenilalanina/análogos & derivados , Fenilalanina/metabolismo , Fenilalanina/toxicidade , Sulfonas/química , Sulfonas/metabolismo , Sulfonas/toxicidadeRESUMO
The importance of the cell wall to the viability of the bacterium is underscored by the breadth of antibiotic structures that act by blocking key enzymes that are tasked with cell-wall creation, preservation, and regulation. The interplay between cell-wall integrity, and the summoning forth of resistance mechanisms to deactivate cell-wall-targeting antibiotics, involves exquisite orchestration among cell-wall synthesis and remodeling and the detection of and response to the antibiotics through modulation of gene regulation by specific effectors. Given the profound importance of antibiotics to the practice of medicine, the assertion that understanding this interplay is among the most fundamentally important questions in bacterial physiology is credible. The enigmatic regulation of the expression of the AmpC ß-lactamase, a clinically significant and highly regulated resistance response of certain Gram-negative bacteria to the ß-lactam antibiotics, is the exemplar of this challenge. This review gives a current perspective to this compelling, and still not fully solved, 35-year enigma.
Assuntos
Parede Celular/metabolismo , Bactérias Gram-Negativas/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Parede Celular/química , Farmacorresistência Bacteriana/efeitos dos fármacos , Glicosiltransferases/química , Glicosiltransferases/metabolismo , Hexosaminidases/química , Hexosaminidases/metabolismo , Proteínas de Ligação às Penicilinas/química , Proteínas de Ligação às Penicilinas/metabolismo , beta-Lactamases/química , beta-Lactamases/metabolismoRESUMO
The lytic transglycosylases (LTs) are bacterial enzymes that catalyze the non-hydrolytic cleavage of the peptidoglycan structures of the bacterial cell wall. They are not catalysts of glycan synthesis as might be surmised from their name. Notwithstanding the seemingly mundane reaction catalyzed by the LTs, their lytic reactions serve bacteria for a series of astonishingly diverse purposes. These purposes include cell-wall synthesis, remodeling, and degradation; for the detection of cell-wall-acting antibiotics; for the expression of the mechanism of cell-wall-acting antibiotics; for the insertion of secretion systems and flagellar assemblies into the cell wall; as a virulence mechanism during infection by certain Gram-negative bacteria; and in the sporulation and germination of Gram-positive spores. Significant advances in the mechanistic understanding of each of these processes have coincided with the successive discovery of new LTs structures. In this review, we provide a systematic perspective on what is known on the structure-function correlations for the LTs, while simultaneously identifying numerous opportunities for the future study of these enigmatic enzymes.
Assuntos
Bactérias/enzimologia , Parede Celular/enzimologia , Glicosiltransferases/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias , Peptidoglicano/metabolismoRESUMO
Lytic transglycosylases (LTs) are bacterial enzymes that catalyze the cleavage of the glycan strands of the bacterial cell wall. The mechanism of this cleavage is a remarkable intramolecular transacetalization reaction, accomplished by an ensemble of active-site residues. Because the LT reaction occurs in parallel with the cell wall bond-forming reactions catalyzed by the penicillin-binding proteins, simultaneous inhibition of both enzymes can be particularly bactericidal to Gram-negative bacteria. The MltE lytic transglycosylase is the smallest of the eight LTs encoded by the Escherichia coli genome. Prior crystallographic and computational studies identified four active-site residues-E64, S73, S75, and Y192-as playing roles in catalysis. Each of these four residues was individually altered by mutation to give four variant enzymes (E64Q, S73A, S75A, and Y192F). All four variants showed reduced catalytic activity [soluble wild type (100%) > soluble Y192F and S75A (both 40%) > S73A (4%) > E64Q (≤1%)]. The crystal structure of each variant protein was determined at the resolution of 2.12 Å for E64Q, 2.33 Å for Y192F, 1.38 Å for S73A, and 1.35 Å for S75A. These variants show alteration of the hydrogen-bond interactions of the active site. Within the framework of a prior computational study of the LT mechanism, we suggest the mechanistic role of these four active-site residues in MltE catalysis.
Assuntos
Escherichia coli K12/enzimologia , Proteínas de Escherichia coli/química , Glicosiltransferases/química , Substituição de Aminoácidos , Catálise , Domínio Catalítico , Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Glicosiltransferases/genética , Mutação de Sentido IncorretoRESUMO
Formation of catenanes by proteins is rare, with few known examples. We report herein the X-ray structure of a catenane dimer of lytic transglycosylase SltB1 of Pseudomonas aeruginosa. The enzyme is soluble and exists in the periplasmic space, where it modifies the bacterial cell wall. The catenane dimer exhibits the protein monomers in a noncovalent chain-link arrangement, whereby a stretch of 51 amino acids (to become a loop and three helices) from one monomer threads through the central opening of the structure of the partner monomer. The protein folds after threading in a manner that leaves two helices (α1 and α2) as stoppers to impart stability to the dimer structure. The symmetric embrace by the two SltB1 molecules occludes both active sites entirely, an arrangement that is sustained by six electrostatic interactions between the two monomers. In light of the observation of these structural motifs in all members of Family 3 lytic transglycosylases, catenanes might be present for those enzymes, as well. The dimeric catenane might represent a regulated form of SltB1.
Assuntos
Cristalografia por Raios X , Peptidoglicano Glicosiltransferase/química , Peptidoglicano Glicosiltransferase/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Modelos Moleculares , Conformação Proteica , Dobramento de ProteínaRESUMO
A complex link exists between cell-wall recycling/repair and the manifestation of resistance to ß-lactam antibiotics in many Enterobacteriaceae and Pseudomonas aeruginosa. This process is mediated by specific cell-wall-derived muropeptide products. These muropeptides are internalized into the cytoplasm and bind to the transcriptional regulator AmpR, which controls the cytoplasmic events that lead to expression of ß-lactamase, an antibiotic-resistance determinant. The effector-binding domain (EBD) of AmpR was purified to homogeneity. We document that the EBD exists exclusively as a dimer, even at a concentration as low as 1 µM. The EBD binds to the suppressor ligand UDP-N-acetyl-ß-d-muramyl-l-Ala-γ-d-Glu-meso-DAP-d-Ala-d-Ala and binds to two activator muropeptides, N-acetyl-ß-d-glucosamine-(1â4)-1,6-anhydro-N-acetyl-ß-d-muramyl-l-Ala-γ-d-Glu-meso-DAP-d-Ala-d-Ala and 1,6-anhydro-N-acetyl-ß-d-muramyl-l-Ala-γ-d-Glu-meso-DAP-d-Ala-d-Ala, as assessed by non-denaturing mass spectrometry. The EBD does not bind to 1,6-anhydro-N-acetyl-ß-d-muramyl-l-Ala-γ-d-Glu-meso-DAP. This binding selectivity revises the dogma in the field. The crystal structure of the EBD dimer was solved to 2.2 Å resolution. The EBD crystallizes in a "closed" conformation, in contrast to the "open" structure required to bind the muropeptides. Structural issues of this ligand recognition are addressed by molecular dynamics simulations, which reveal significant differences among the complexes with the effector molecules.
Assuntos
Proteínas de Bactérias/química , Peptídeos/química , Pseudomonas aeruginosa/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Peptídeos/metabolismo , Domínios Proteicos , Pseudomonas aeruginosa/metabolismoRESUMO
The major constituent of bacterial cell walls is peptidoglycan, which, in its crosslinked form, is a polymer of considerable complexity that encases the entire bacterium. A functional cell wall is indispensable for survival of the organism. There are several dozen enzymes that assemble and disassemble the peptidoglycan dynamically within each bacterial generation. Understanding of the nature of these transformations is critical knowledge for these events. Octasaccharide peptidoglycans were prepared and studied with seven recombinant cell-wall-active enzymes (SltB1, MltB, RlpA, mutanolysin, AmpDh2, AmpDh3, and PBP5). With the use of highly sensitive mass spectrometry methods, we described the breadth of reactions that these enzymes catalyzed with peptidoglycan and shed light on the nature of the cell wall alteration performed by these enzymes. The enzymes exhibit broadly distinct preferences for their substrate peptidoglycans in the reactions that they catalyze.
Assuntos
Bactérias/metabolismo , Parede Celular/metabolismo , Enzimas/metabolismo , Biocatálise , Cromatografia Líquida de Alta Pressão , Endopeptidases/genética , Endopeptidases/metabolismo , Enzimas/genética , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Espectrometria de Massas , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Peptidoglicano/análise , Peptidoglicano/química , Peptidoglicano/metabolismo , Pseudomonas aeruginosa/enzimologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Streptomyces griseus/enzimologia , Especificidade por Substrato , Transferases/genética , Transferases/metabolismoRESUMO
An enzyme superfamily, the lytic transglycosylases (LTs), occupies the space between the two membranes of Gram-negative bacteria. LTs catalyze the non-hydrolytic cleavage of the bacterial peptidoglycan cell-wall polymer. This reaction is central to the growth of the cell wall, for excavating the cell wall for protein insertion, and for monitoring the cell wall so as to initiate resistance responses to cell-wall-acting antibiotics. The nefarious Gram-negative pathogen Pseudomonas aeruginosa encodes eleven LTs. With few exceptions, their substrates and functions are unknown. Each P. aeruginosa LT was expressed as a soluble protein and evaluated with a panel of substrates (both simple and complex mimetics of their natural substrates). Thirty-one distinct products distinguish these LTs with respect to substrate recognition, catalytic activity, and relative exolytic or endolytic ability. These properties are foundational to an understanding of the LTs as catalysts and as antibiotic targets.
Assuntos
Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Proteoma/genética , Proteoma/metabolismo , Pseudomonas aeruginosa/enzimologia , Biocatálise , Parede Celular/química , Parede Celular/metabolismo , Conformação Molecular , Pseudomonas aeruginosa/citologiaRESUMO
Systems that perform continuous hypermutation of designated genes without compromising the integrity of the host genome can dramatically accelerate the evolution of new or enhanced protein functions. We describe an orthogonal DNA replication system in E. coli based on the controlled expression of the replisome of bacteriophage T7. The system replicates circular plasmids that enable high transformation efficiencies and seamless integration into standard molecular biology workflows. Engineering of T7 DNA polymerase yielded variant proteins with mutation rates of 1.7 × 10 -5 substitutions per base in vivo - 100,000-fold above the genomic mutation rate. Continuous evolution using the mutagenic T7 replisome was demonstrated by expanding the substrate scope of TEM-1 ß-lactamase and increase activity 1,000-fold against clinically relevant monobactam and cephalosporin antibiotics in less than one week.
RESUMO
Live vaccines are ideal for inducing immunity but suffer from the need to attenuate their pathogenicity or replication to preclude the possibility of escape. Unnatural amino acids (UAAs) provide a strategy to engineer stringent auxotrophies, yielding conditionally replication incompetent live bacteria with excellent safety profiles. Here, we engineer Pseudomonas aeruginosa to maintain auxotrophy for the UAA p-benzoyl-L-phenylalanine (BzF) through its incorporation into the essential protein DnaN. In vivo evolution using an Escherichia coli-based two-hybrid selection system enabled engineering of a mutant DnaN homodimeric interface completely dependent on a BzF-specific interaction. This engineered strain, Pa Vaccine, exhibits undetectable escape frequency (<10-11) and shows excellent safety in naïve mice. Animals vaccinated via intranasal or intraperitoneal routes are protected from lethal challenge with pathogenic P. aeruginosa PA14. These results establish UAA-auxotrophic bacteria as promising candidates for bacterial vaccine therapy and outline a platform for expanding this technology to diverse bacterial pathogens.
Assuntos
Infecções por Pseudomonas , Vacinas contra Pseudomonas , Pseudomonas aeruginosa , Animais , Pseudomonas aeruginosa/imunologia , Pseudomonas aeruginosa/genética , Infecções por Pseudomonas/prevenção & controle , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/microbiologia , Camundongos , Feminino , Vacinas contra Pseudomonas/imunologia , Vacinas contra Pseudomonas/genética , Aminoácidos , Fenilalanina/análogos & derivados , Escherichia coli/imunologia , Escherichia coli/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/genética , Camundongos Endogâmicos BALB CRESUMO
The maturation of chemical synthesis during the 20th century has elevated the discipline from a largely empirical into a rational science. This ability to purposefully craft matter at the molecular level has put chemists in a privileged position to contribute to progress in neighboring natural sciences. Recently, we have witnessed another major advance in the field in which chemists use chemical and biological "synthetic" methods together to alter the structures and properties of biological macromolecules in ways heretofore unimagined. This interdisciplinary approach to synthesis has even allowed us to expand upon the defining characteristics of living organisms at the molecular level. In this perspective, we present a case study for the successful addition of new chemistries to the fundamental processes of the central dogma of molecular biology, exemplified by the expansion of the genetic code.
RESUMO
Gram-negative bacteria have evolved an elaborate pathway to sense and respond to exposure to ß-lactam antibiotics. The ß-lactam antibiotics inhibit penicillin-binding proteins, whereby the loss of their activities alters/damages the cell-wall peptidoglycan. Bacteria sense this damage and remove the affected peptidoglycan into complex recycling pathways. As an offshoot of these pathways, muropeptide chemical signals generated from the cell-wall recycling manifest the production of a class C ß-lactamase, which hydrolytically degrades the ß-lactam antibiotic as a resistance mechanism. We disclose the use of a fluorescence probe that detects the activation of the recycling system by the formation of the key muropeptides involved in signaling. This same probe additionally detects natural-product cell-wall-active antibiotics that are produced in situ by cohabitating bacteria.
Assuntos
Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Corantes Fluorescentes/química , Imagem Óptica/métodos , Pseudomonas aeruginosa/efeitos dos fármacos , beta-Lactamas/metabolismo , Produtos Biológicos/metabolismo , Parede Celular/metabolismo , Metaboloma/efeitos dos fármacos , Proteínas de Ligação às Penicilinas/metabolismo , Transdução de Sinais , Resistência beta-Lactâmica/efeitos dos fármacosRESUMO
SPOR domains are widely present in bacterial proteins that recognize cell-wall peptidoglycan strands stripped of the peptide stems. This type of peptidoglycan is enriched in the septal ring as a product of catalysis by cell-wall amidases that participate in the separation of daughter cells during cell division. Here, we document binding of synthetic denuded glycan ligands to the SPOR domain of the lytic transglycosylase RlpA from Pseudomonas aeruginosa (SPOR-RlpA) by mass spectrometry and structural analyses, and demonstrate that indeed the presence of peptide stems in the peptidoglycan abrogates binding. The crystal structures of the SPOR domain, in the apo state and in complex with different synthetic glycan ligands, provide insights into the molecular basis for recognition and delineate a conserved pattern in other SPOR domains. The biological and structural observations presented here are followed up by molecular-dynamics simulations and by exploration of the effect on binding of distinct peptidoglycan modifications.
Assuntos
Parede Celular/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Peptidoglicano/química , Domínios Proteicos , Bacillus subtilis/química , Bacillus subtilis/metabolismo , Sequência de Carboidratos , Parede Celular/metabolismo , Cristalografia por Raios X , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Lipoproteínas/química , Lipoproteínas/metabolismo , Simulação de Dinâmica Molecular , Peptidoglicano/metabolismo , Ligação Proteica , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/metabolismoRESUMO
The interplay between the activities of lytic transglycosylases (LTs) and penicillin-binding proteins (PBPs) is critical for the health of the bacterial cell wall. Bulgecin A (a natural-product inhibitor of LTs) potentiates the activity of ß-lactam antibiotics (inhibitors of PBPs), underscoring this intimate mechanistic interdependence. Bulgecin A in the presence of an appropriate ß-lactam causes bulge deformation due to the formation of aberrant peptidoglycan at the division site of the bacterium. As Pseudomonas aeruginosa, a nefarious human pathogen, has 11 LT paralogs, the answer as to which LT activity correlates with ß-lactam potentiation is important and is currently unknown. Growth of P. aeruginosa PAO1 strains harboring individual transposon-insertion mutants at each of the 11 genes for LTs, in the presence of the ß-lactam antibiotic ceftazidime or meropenem, implicated the gene products of slt, mltD, and mltG (of the 11), in bulge formation and potentiation. Hence, the respective enzymes would be the targets of inhibition by bulgecin A, which was indeed documented. We further demonstrated by imaging in real time and by SEM that cell lysis occurs by the structural failure of this bulge. Upon removal of the ß-lactam antibiotic prior to lysis, P. aeruginosa experiences delayed recovery from the elongation and bulge phenotype in the presence of bulgecin A. These observations argue for a collaborative role for the target LTs in the repair of the aberrant cell wall, the absence of activities of which in the presence of bulgecin A results in potentiation of the ß-lactam antibiotic.
Assuntos
Acetilglucosamina/análogos & derivados , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Prolina/análogos & derivados , Pseudomonas aeruginosa/efeitos dos fármacos , beta-Lactamas/farmacologia , Acetilglucosamina/farmacologia , Testes de Sensibilidade Microbiana , Prolina/farmacologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/fisiologiaRESUMO
The bulgecins are iminosaccharide secondary metabolites of the Gram-negative bacterium Paraburkholderia acidophila and inhibitors of lytic transglycosylases of bacterial cell-wall biosynthesis and remodeling. The activities of the bulgecins are intimately intertwined with the mechanism of a cobiosynthesized ß-lactam antibiotic. ß-Lactams inhibit the penicillin-binding proteins, enzymes also critical to cell-wall biosynthesis. The simultaneous loss of the lytic transglycosylase (by bulgecin) and penicillin-binding protein (by ß-lactams) activities results in deformation of the septal cell wall, observed microscopically as a bulge preceding bacterial cell lysis. We describe a practical synthesis of the three naturally occurring bulgecin iminosaccharides and their mechanistic evaluation in a series of microbiological studies. These studies identify potentiation by the bulgecin at subminimum inhibitory concentrations of the ß-lactam against three pathogenic Gram-negative bacteria and establish for the first time that this potentiation results in a significant increase in the bactericidal efficacy of a clinical ß-lactam.
Assuntos
Glicopeptídeos/síntese química , Glicopeptídeos/farmacologia , Inibidores de beta-Lactamases/síntese química , Inibidores de beta-Lactamases/farmacologia , beta-Lactamas/síntese química , beta-Lactamas/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Antibacterianos/farmacologia , Técnicas de Química Sintética , Relação Dose-Resposta a Droga , Glicopeptídeos/química , Testes de Sensibilidade Microbiana , Modelos Biológicos , Estrutura Molecular , Inibidores de beta-Lactamases/química , beta-Lactamas/químicaRESUMO
Lytic transglycosylases (LTs) catalyze the non-hydrolytic cleavage of the bacterial cell wall by an intramolecular transacetalization reaction. This reaction is critically and broadly important in modifications of the bacterial cell wall in the course of its biosynthesis, recycling, manifestation of virulence, insertion of structural entities such as the flagellum and the pili, among others. The first QM/MM analysis of the mechanism of reaction of an LT, that for the Escherichia coli MltE, is undertaken. The study reveals a conformational itinerary consistent with an oxocarbenium-like transition state, characterized by a pivotal role for the active-site glutamic acid in proton transfer. Notably, an oxazolinium intermediate, as a potential intermediate, is absent. Rather, substrate-assisted catalysis is observed through a favorable dipole provided by the N-acetyl carbonyl group of MurNAc saccharide. This interaction stabilizes the incipient positive charge development in the transition state. This mechanism coincides with near-synchronous acetal cleavage and acetal formation.
Assuntos
Parede Celular/enzimologia , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Glicosiltransferases/química , Modelos Moleculares , Sistemas de Secreção Tipo VI/química , Domínio Catalítico , Proteínas de Escherichia coli/metabolismo , Glicosiltransferases/metabolismo , Sistemas de Secreção Tipo VI/metabolismoRESUMO
Bacteria grow and divide without loss of cellular integrity. This accomplishment is notable, as a key component of their cell envelope is a surrounding glycopeptide polymer. In Gram-negative bacteria this polymer-the peptidoglycan-grows by the difference between concurrent synthesis and degradation. The regulation of the enzymatic ensemble for these activities is poorly understood. We report herein the structural basis for the control of one such enzyme, the lytic transglycosylase MltF of Pseudomonas aeruginosa. Its structure comprises two modules: an ABC-transporter-like regulatory module and a catalytic module. Occupancy of the regulatory module by peptidoglycan-derived muropeptides effects a dramatic and long-distance (40 Å) conformational change, occurring over the entire protein structure, to open its active site for catalysis. This discovery of the molecular basis for the allosteric control of MltF catalysis is foundational to further study of MltF within the complex enzymatic orchestration of the dynamic peptidoglycan.