RESUMO
Cyclopropane fatty acid synthases (CFAS) catalyze the conversion of unsaturated fatty acids to cyclopropane fatty acids (CFAs) within bacterial membranes. This modification alters the biophysical properties of membranes and has been correlated with virulence in several human pathogens. Despite the central role played by CFAS enzymes in regulating bacterial stress responses, the mechanistic properties of the CFAS enzyme family and the consequences of CFA biosynthesis remain largely uncharacterized in most bacteria. We report the first characterization of the CFAS enzyme from Pseudomonas aeruginosa (PA), an opportunistic human pathogen with complex membrane biology that is frequently associated with antimicrobial resistance and high tolerance to various external stressors. We demonstrate that CFAs are produced by a single enzyme in PA and that cfas gene expression is upregulated during the transition to stationary phase and in response to oxidative stress. Analysis of PA lipid extracts reveal a massive increase in CFA production as PA cells enter stationary phase and help define the optimal membrane composition for in vitro assays. The purified PA-CFAS enzyme forms a stable homodimer and preferentially modifies phosphatidylglycerol lipid substrates and membranes with a higher content of unsaturated acyl chains. Bioinformatic analysis across bacterial phyla shows highly divergent amino acid sequences within the lipid-binding domain of CFAS enzymes, perhaps suggesting distinct membrane-binding properties among different orthologs. This work lays an important foundation for further characterization of CFAS in P. aeruginosa and for examining the functional differences between CFAS enzymes from different bacteria.
RESUMO
Biofilm infections are a major cause of food poisoning and hospital-acquired infections. Quaternary ammonium compounds are a group of effective disinfectants widely used in industry and households, yet their efficacy is lessened when used as antibiofilm agents compared to that against planktonic bacteria. It is therefore necessary to identify alternative formulations of quaternary ammonium compounds to achieve an effective biofilm dispersal. Quaternary ammonium amphiphiles can form vesicular structures termed "quatsomes" in the presence of cholesterol. In addition to their intrinsic antimicrobial properties, quatsomes can also be used for the delivery of other types of antibiotics or biomarkers. In this study, quatsomes were prepared from binary mixtures of cholesterol and mono- or dialkyl-quaternary ammonium compounds; then, the integrity and stability of their vesicular structure were assessed and related to monomer chain number and chain length. The quatsomes were used to treat Pseudomonas aeruginosa biofilms, showing effective antibiofilm abilities comparable to those of their monomers. A systematic liquid chromatography-mass spectrometry method for quantifying quatsome vesicle components was also developed and used to establish the significance of cholesterol in the quatsome self-assembly processes.
Assuntos
Anti-Infecciosos , Pseudomonas aeruginosa , Compostos de Amônio Quaternário , Biofilmes , Antibacterianos/farmacologia , Colesterol , Testes de Sensibilidade MicrobianaRESUMO
Inspired largely by the role of the posttranslationally modified amino acid dopa (DOPA) in mussel adhesion, catechol functional groups have become commonplace in medical adhesives, tissue scaffolds, and advanced smart polymers. Yet, the complex redox chemistry of catechol groups complicates cross-link regulation, hampering fabrication and the long-term stability/performance of mussel-inspired polymers. Here, we investigated the various fates of DOPA residues in proteins comprising mussel byssus fibers before, during, and after protein secretion. Utilizing a combination of histological staining and confocal Raman spectroscopy on native tissues, as well as peptide-based cross-linking studies, we have identified at least two distinct DOPA-based cross-linking pathways during byssus fabrication, achieved by oxidative covalent cross-linking or formation of metal coordination interactions under reducing conditions, respectively. We suggest that these end states are spatiotemporally regulated by the microenvironments in which the proteins are stored prior to secretion, which are retained after formation-in particular, due to the presence of reducing moieties. These findings provide physicochemical pathways toward greater control over properties of synthetic catechol-based polymers and adhesives.
Assuntos
Bivalves/metabolismo , Catecóis/metabolismo , Di-Hidroxifenilalanina/metabolismo , Sequência de Aminoácidos , Animais , Catecóis/química , Oxirredução , Peptídeos/química , Peptídeos/metabolismoRESUMO
Class II lanthipeptide synthetases (LanM enzymes) catalyze the installation of multiple thioether bridges into genetically encoded peptides to produce macrocyclic lanthipeptides, a class of biologically active natural products. Collectively, LanM enzymes install thioether rings of different sizes, topologies, and stereochemistry into a vast array of different LanA precursor peptide sequences. The factors that govern the outcome of the LanM-catalyzed reaction cascade are not fully characterized but are thought to involve both intermolecular interactions and intramolecular conformational changes in the [LanM:LanA] Michaelis complex. To test this hypothesis, we have combined AlphaFold modeling with hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis of a small collection of divergent LanM/LanA systems to investigate the similarities and differences in their conformational dynamic properties. Our data indicate that LanA precursor peptide binding triggers relatively conserved changes in the structural dynamics of the LanM dehydratase domain, supporting the existence of a similar leader peptide binding mode across the LanM family. In contrast, changes induced in the dynamics of the LanM cyclase domain were more highly variable between enzymes, perhaps reflecting different peptide-cyclase interactions and/or different modes of allosteric activation in class II lanthipeptide biosynthesis. Our analysis highlights the ability of the emerging AlphaFold platform to predict protein-peptide interactions that are supported by other lines of experimental evidence. The combination of AlphaFold modeling with HDX-MS analysis should emerge as a useful approach for investigating other conformationally dynamic enzymes involved in peptide natural product biosynthesis.
Assuntos
Produtos Biológicos , Espectrometria de Massa com Troca Hidrogênio-Deutério , Deutério , Medição da Troca de Deutério , Hidroliases/metabolismo , Ligases/metabolismo , Peptídeos/química , Sinais Direcionadores de Proteínas , SulfetosRESUMO
Lanthipeptide synthetases construct macrocyclic peptide natural products by catalyzing an iterative cascade of post-translational modifications. Class II lanthipeptide synthetases (LanM enzymes) catalyze multiple rounds of peptide dehydration and thioether macrocycle formation in a manner that guides precursor peptide maturation to the biologically active final product with high fidelity. The mechanistic details underlying the contradictory phenomena of substrate flexibility coupled with high biosynthetic fidelity have proven challenging to illuminate. In this work, we employ mass spectrometry to investigate how the structure of a maturing precursor lanthipeptide (HalA2) influences the local and global structure of its cognate lanthipeptide synthetase (HalM2). Using enzymatically synthesized HalA2 peptides that contain sets of native thioether macrocycles, we employ ion mobility mass spectrometry (IM-MS) to show that HalA2 macrocyclization alters the conformational landscape of the HalM2 enzyme in a systematic manner. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) studies show that local HalM2 structural dynamics also change in response to HalA2 post-translational modification. Notably, deuterium uptake in a critical HalM2 α-helical region depends on the number of thioether macrocycles present in the HalA2 core peptide. Binding of the isolated leader and core peptide portions of the modular HalA2 precursor led to a synergistic structuring of this α-helical region, providing evidence for distinct leader and core peptide binding sites that independently alter the dynamics of this functionally critical α-helix. The data support a mechanistic model where the sequential post-translational modification of HalA2 alters the conformational dynamics of HalM2 in regions of the enzyme that are known to be functionally critical.
Assuntos
Bacteriocinas , Ligases , Bacteriocinas/química , Deutério , Ligases/metabolismo , Peptídeos , SulfetosRESUMO
Lanthipeptides are ribosomally synthesized and post-translationally modified peptide (RiPP) natural products. These genetically encoded peptides are biosynthesized by multifunctional enzymes (lanthipeptide synthetases) that possess relaxed substrate specificity and catalyze iterative rounds of post-translational modification. Recent evidence has suggested that some lanthipeptide synthetases are structurally dynamic enzymes that are allosterically activated by precursor peptide binding and that conformational sampling of the enzyme-peptide complex may play an important role in defining the efficiency and sequence of biosynthetic events. These "biophysical" processes, while critical for defining the activity and function of the synthetase, remain very challenging to study with existing methodologies. Herein, we show that native mass spectrometry coupled to ion mobility (native IM-MS) provides a powerful and sensitive means for investigating the conformational landscapes and intermolecular interactions of lanthipeptide synthetases. Namely, we demonstrate that the class II lanthipeptide synthetase (HalM2) and its noncovalent complex with the cognate HalA2 precursor peptide can be delivered into the gas phase in a manner that preserves native structures and intermolecular enzyme-peptide contacts. Moreover, gas phase ion mobility studies of the natively folded ions demonstrate that peptide binding and mutations to dynamic structural elements of HalM2 alter the conformational landscape of the enzyme. Cumulatively, these data support previous claims that lanthipeptide synthetases are structurally dynamic enzymes that undergo functionally relevant conformational changes in response to precursor peptide binding. This work establishes native IM-MS as a versatile approach for characterizing intermolecular interactions and for unraveling the relationships between protein structure and biochemical function in RiPP biosynthetic systems.
Assuntos
Bacteriocinas/metabolismo , Espectrometria de Mobilidade Iônica/métodos , Ligases/metabolismo , Sequência de Aminoácidos , Bacteriocinas/química , Catálise , Ligases/química , Espectrometria de Massas/métodos , Conformação Molecular , Peptídeos/química , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Ribossomos/metabolismo , Especificidade por SubstratoRESUMO
Cytochrome P450 3A4 (CYP3A4) is the most important drug-metabolizing enzyme in humans and has been associated with harmful drug interactions. The activity of CYP3A4 is known to be modulated by several compounds and by the electron transfer partner, cytochrome P450 reductase (CPR). The underlying mechanism of these effects, however, is poorly understood. We have used hydrogen-deuterium exchange mass spectrometry to investigate the impact of binding of CPR and of three different substrates (7-benzyloxy-4-trifluoromethyl-coumarin, testosterone, and progesterone) on the conformational dynamics of CYP3A4. Here, we report that interaction of CYP3A4 with substrates or with the oxidized or reduced forms of CPR leads to a global rigidification of the CYP3A4 structure. This was evident from the suppression of deuterium exchange in several regions of CYP3A4, including regions known to be involved in protein-protein interactions (helix C) and substrate binding and specificity (helices B' and E, and loop K/ß1). Furthermore, the bimodal isotopic distributions observed for some CYP3A4-derived peptides were drastically impacted upon binding to CPR and/or substrates, suggesting the existence of stable CYP3A4 conformational populations that are perturbed by ligand/CPR binding. The results have implications for understanding the mechanisms of ligand binding, allostery, and catalysis in CYP enzymes.
Assuntos
Citocromo P-450 CYP3A/metabolismo , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Animais , Citocromo P-450 CYP3A/química , Humanos , Modelos Moleculares , NADPH-Ferri-Hemoproteína Redutase/química , Ligação Proteica , Conformação Proteica , Mapas de Interação de Proteínas , Ratos , Especificidade por SubstratoRESUMO
Class II lanthipeptide synthetases (LanM enzymes) catalyze the multistep post-translational modification of genetically encoded precursor peptides into macrocyclic (often antimicrobial) lanthipeptides. The reaction sequence involves dehydration of serine/threonine residues, followed by intramolecular addition of cysteine thiols onto the nascent dehydration sites to construct thioether bridges. LanMs utilize two separate active sites in an iterative yet highly coordinated manner to maintain a remarkable level of regio- and stereochemical control over the multistep maturation. The mechanisms underlying this biosynthetic fidelity remain enigmatic. We recently demonstrated that proper function of the haloduracin ß synthetase (HalM2) requires dynamic structural elements scattered across the surface of the enzyme. Here, we perform kinetic simulations, structural analysis of reaction intermediates, hydrogen-deuterium exchange mass spectrometry studies, and molecular dynamics simulations to investigate the contributions of these dynamic HalM2 structural elements to biosynthetic efficiency and fidelity. Our studies demonstrate that a large, conserved loop (HalM2 residues P349-P405) plays essential roles in defining the precursor peptide binding site, facilitating efficient peptide dehydration, and guiding the order of thioether ring formation. Moreover, mutations near the interface of the HalM2 dehydratase and cyclase domains perturb cyclization fidelity and result in aberrant thioether topologies that cannot be corrected by the wild type enzyme, suggesting an element of kinetic control in the normal cyclization sequence. Overall, this work provides the most comprehensive correlation of the structural and functional properties of a LanM enzyme reported to date and should inform mechanistic studies of the biosynthesis of other ribosomally synthesized and post-translationally modified peptide natural products.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Bacteriocinas/química , Ligases/química , Sequência de Aminoácidos/genética , Bacteriocinas/metabolismo , Sítios de Ligação/genética , Ciclização , Espectrometria de Massa com Troca Hidrogênio-Deutério/métodos , Cinética , Ligases/metabolismo , Mutação/genética , Peptídeos/química , Processamento de Proteína Pós-Traducional/genética , Ribossomos/metabolismo , Especificidade por Substrato/genéticaRESUMO
The biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs) proceeds via the multistep maturation of genetically encoded precursor peptides, often catalyzed by enzymes with multiple functions and iterative activities. Recent studies have suggested that, among other factors, conformational sampling of enzyme:peptide complexes likely plays a critical role in defining the kinetics and, ultimately, the set of post-translational modifications in these systems. However, detailed characterizations of these putative conformational sampling mechanisms have not yet been possible on many RiPP biosynthetic systems. In this study, we report the first comprehensive application of hydrogen-deuterium exchange mass spectrometry (HDX-MS) to study the biophysical properties of a RiPP biosynthetic enzyme. Using the well-characterized class II lanthipeptide synthetase HalM2 as a model system, we have employed HDX-MS to demonstrate that HalM2 is indeed a highly structurally dynamic enzyme. Using this HDX-MS approach, we have identified novel precursor peptide binding elements, have uncovered long-range structural communication across the enzyme that is triggered by ligand binding and ATP hydrolysis, and have detected specific interactions between the HalM2 synthetase and the leader- and core-peptide subdomains of the modular HalA2 precursor peptide substrate. The functional relevance of the dynamic HalM2 elements discovered in this study are validated with biochemical assays and kinetic analysis of a panel of HDX-MS guided variant enzymes. Overall, the data have provided a wealth of fundamentally new information on LanM systems that will inform the rational manipulation and engineering of these impressive multifunctional catalysts. Moreover, this work highlights the broad utility of the HDX-MS platform for revealing important biophysical properties and enzyme structural dynamics that likely play a widespread role in RiPP biosynthesis.
Assuntos
Espectrometria de Massa com Troca Hidrogênio-Deutério/métodos , Elementos da Série dos Lantanídeos/química , Peptídeo Sintases/química , Trifosfato de Adenosina/metabolismo , Adenilil Imidodifosfato/metabolismo , Sequência de Aminoácidos , Fenômenos Biofísicos , Hidrogênio/química , Hidrólise , Ligantes , Peptídeo Sintases/metabolismo , Ligação Proteica , Conformação Proteica , Especificidade por SubstratoRESUMO
Although natural products have been a particularly rich source of human medicines, activity-based screening results in a very high rate of rediscovery of known molecules. Based on the large number of natural product biosynthetic genes in microbial genomes, many have proposed "genome mining" as an alternative approach for discovery efforts; however, this idea has yet to be performed experimentally on a large scale. Here, we demonstrate the feasibility of large-scale, high-throughput genome mining by screening a collection of over 10,000 actinomycetes for the genetic potential to make phosphonic acids, a class of natural products with diverse and useful bioactivities. Genome sequencing identified a diverse collection of phosphonate biosynthetic gene clusters within 278 strains. These clusters were classified into 64 distinct groups, of which 55 are likely to direct the synthesis of unknown compounds. Characterization of strains within five of these groups resulted in the discovery of a new archetypical pathway for phosphonate biosynthesis, the first (to our knowledge) dedicated pathway for H-phosphinates, and 11 previously undescribed phosphonic acid natural products. Among these compounds are argolaphos, a broad-spectrum antibacterial phosphonopeptide composed of aminomethylphosphonate in peptide linkage to a rare amino acid N(5)-hydroxyarginine; valinophos, an N-acetyl l-Val ester of 2,3-dihydroxypropylphosphonate; and phosphonocystoximate, an unusual thiohydroximate-containing molecule representing a new chemotype of sulfur-containing phosphonate natural products. Analysis of the genome sequences from the remaining strains suggests that the majority of the phosphonate biosynthetic repertoire of Actinobacteria has been captured at the gene level. This dereplicated strain collection now provides a reservoir of numerous, as yet undiscovered, phosphonate natural products.
Assuntos
Actinobacteria/química , Actinobacteria/genética , Produtos Biológicos/química , Descoberta de Drogas/tendências , Genoma Bacteriano/genética , Genômica/métodos , Ácidos Fosforosos/análise , Sequência de Bases , Descoberta de Drogas/métodos , Biblioteca Gênica , Genômica/tendências , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
The chemical versatility of the flavin coenzyme is nearly unparalleled in enzyme catalysis. An interesting illustration of this versatility can be found in the reaction catalyzed by the type II isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI-2) - an enzyme that interconverts the two essential isoprene units (isopentenyl pyrophosphate and dimethylallyl pyrophosphate) that are needed to initiate the biosynthesis of all isoprenoids. Over the past decade, a variety of biochemical, spectroscopic, structural and mechanistic studies of IDI-2 have provided mounting evidence that the flavin coenzyme of IDI-2 acts in a most unusual manner - as an acid/base catalyst to mediate a 1,3-proton addition/elimination reaction. While not entirely without precedent, IDI-2 is by far the most extensively studied flavoenzyme that employs flavin-mediated acid/base catalysis. Thus, IDI-2 serves as an important mechanistic model for understanding this often overlooked, but potentially widespread reactivity of flavin coenzymes. This review details the most pertinent studies that have contributed to the development of mechanistic proposals for this highly unusual flavoenzyme, and discusses future experiments that may be able to clarify remaining uncertainties in the chemical mechanism of IDI-2.
Assuntos
Isomerases de Ligação Dupla Carbono-Carbono/química , Flavoproteínas/química , Isomerases de Ligação Dupla Carbono-Carbono/metabolismo , Flavoproteínas/metabolismo , Hemiterpenos/química , Hemiterpenos/metabolismo , Compostos Organofosforados/química , Compostos Organofosforados/metabolismo , Terpenos/química , Terpenos/metabolismoRESUMO
The mechanisms by which lanthipeptide synthetases control the order in which they catalyze multiple chemical processes are poorly understood. The lacticin 481 synthetase (LctM) cleaves eight chemical bonds and forms six new chemical bonds in a controlled and ordered process. Two general mechanisms have been suggested for the temporal and spatial control of these transformations. In the spatial positioning model, leader peptide binding promotes certain reactions by establishing the spatial orientation of the substrate peptide relative to the synthetase active sites. In the intermediate structure model, the LctM-catalyzed dehydration and cyclization reactions that occur in two distinct active sites orchestrate the overall process by imparting a specific structure into the maturing peptide that facilitates the ensuing reaction. Using isotopically labeled LctA analogues with engineered lacticin 481 biosynthetic machinery and mass spectrometry analysis, we show here that the LctA leader peptide plays critical roles in establishing the modification order and enhancing the catalytic efficiency and fidelity of the synthetase. The data are most consistent with a mechanistic model for LctM where both spatial positioning and intermediate structure contribute to efficient biosynthesis.
Assuntos
Bacteriocinas/biossíntese , Sinais Direcionadores de Proteínas , Água/química , Bacteriocinas/química , CiclizaçãoRESUMO
Lanthipeptides are a class of ribosomally synthesized and posttranslationally modified peptide natural products (RiPPs) that typically harbor multiple intramolecular thioether linkages. For class II lanthipeptides, these cross-links are installed in a multistep reaction pathway by a single enzyme (LanM). The multifunctional nature of LanMs and the manipulability of their genetically encoded peptide substrates (LanAs) make LanM/LanA systems promising targets for the engineering of new antibacterial compounds. Here, we report the development of a semiquantitative mass spectrometry-based assay for kinetic characterization of LanM-catalyzed reactions. The assay was used to conduct a comparative kinetic analysis of two LanM enzymes (HalM2 and ProcM) that exhibit drastically different substrate selectivity. Numerical simulation of the kinetic data was used to develop models for the multistep HalM2- and ProcM-catalyzed reactions. These models illustrate that HalM2 and ProcM have markedly different catalytic efficiencies for the various reactions they catalyze. HalM2, which is responsible for the biosynthesis of a single compound (the Halß subunit of the lantibiotic haloduracin), catalyzes reactions with higher catalytic efficiency than ProcM, which modifies 29 different ProcA precursor peptides during prochlorosin biosynthesis. In particular, the rates of thioether ring formation are drastically reduced in ProcM, likely because this enzyme is charged with installing a variety of lanthipeptide ring architectures in its prochlorosin products. Thus, ProcM appears to pay a kinetic price for its relaxed substrate specificity. In addition, our kinetic models suggest that conformational sampling of the LanM/LanA Michaelis complex could play an important role in the kinetics of LanA maturation.
Assuntos
Alanina/análogos & derivados , Ligases/química , Ligases/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Sulfetos/química , Alanina/química , Bioensaio , Cinética , Ligases/classificação , Modelos Moleculares , Estrutura Molecular , Processamento de Proteína Pós-Traducional , Especificidade por SubstratoRESUMO
The 3C-like protease (3CLpro ) is crucial to the replication of SARS-CoV-2, the causative agent of COVID-19, and is the target of several successful drugs including Paxlovid and Xocova. Nevertheless, the emergence of viral resistance underlines the need for alternative drug strategies. 3CLpro only functions as a homodimer, making the protein-protein interface an attractive drug target. Dimerization is partly mediated by a conserved glycine at position 11. However, some naturally occurring SARS-CoV-2 sequences contain a serine at this position, potentially disrupting the dimer. We have used concentration-dependent activity assays and mass spectrometry to show that indeed the G11S mutation reduces the stability of the dimer by 600-fold. This helps to set a quantitative benchmark for the minimum potency required of any future protein-protein interaction inhibitors targeting 3CLpro and raises interesting questions regarding how coronaviruses bearing such weakly dimerizing 3CLpro enzymes are capable of replication.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Peptídeo Hidrolases/genética , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Mutação , Antivirais/químicaRESUMO
UDP-galactopyranose mutase (UGM) requires reduced FAD (FAD(red)) to catalyze the reversible interconversion of UDP-galactopyranose (UDP-Galp) and UDP-galactofuranose (UDP-Galf). Recent structural and mechanistic studies of UGM have provided evidence for the existence of an FAD-Galf/p adduct as an intermediate in the catalytic cycle. These findings are consistent with Lewis acid/base chemistry involving nucleophilic attack by N5 of FAD(red) at C1 of UDP-Galf/p. In this study, we employed a variety of FAD analogues to characterize the role of FAD(red) in the UGM catalytic cycle using positional isotope exchange (PIX) and linear free energy relationship studies. PIX studies indicated that UGM reconstituted with 5-deaza-FAD(red) is unable to catalyze PIX of the bridging C1-OP(ß) oxygen of UDP-Galp, suggesting a direct role for the FAD(red) N5 atom in this process. In addition, analysis of kinetic linear free energy relationships of k(cat) versus the nucleophilicity of N5 of FAD(red) gave a slope of ρ = -2.4 ± 0.4. Together, these findings are most consistent with a chemical mechanism for UGM involving an S(N)2-type displacement of UDP from UDP-Galf/p by N5 of FAD(red).
Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Flavina-Adenina Dinucleotídeo/análogos & derivados , Flavina-Adenina Dinucleotídeo/química , Transferases Intramoleculares/química , Catálise , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Flavina-Adenina Dinucleotídeo/genética , Flavina-Adenina Dinucleotídeo/metabolismo , Transferases Intramoleculares/genética , Transferases Intramoleculares/metabolismo , CinéticaRESUMO
The enzymes involved in the biosynthesis of carbohydrates and the attachment of sugar units to biological acceptor molecules catalyse an array of chemical transformations and coupling reactions. In prokaryotes, both common sugar precursors and their enzymatically modified derivatives often become substituents of biologically active natural products through the action of glycosyltransferases. Recently, researchers have begun to harness the power of these biological catalysts to alter the sugar structures and glycosylation patterns of natural products both in vivo and in vitro. Biochemical and structural studies of sugar biosynthetic enzymes and glycosyltransferases, coupled with advances in bioengineering methodology, have ushered in a new era of drug development.
Assuntos
Produtos Biológicos/química , Produtos Biológicos/metabolismo , Carboidratos/biossíntese , Produtos Biológicos/biossíntese , Evolução Molecular , Glicosiltransferases/química , Glicosiltransferases/metabolismoRESUMO
Lanthipeptide synthetases are fascinating biosynthetic enzymes that install intramolecular thioether bridges into genetically encoded peptides, typically endowing the peptide with therapeutic properties. The factors that control the macrocyclic topology of lanthipeptides are numerous and remain difficult to predict and manipulate. The key challenge in this endeavor derives from the vast conformational space accessible to the disordered precursor lanthipeptide, which can be manipulated in subtle ways by interaction with the cognate synthetase. This review explores the unique strategies employed by each of the five phylogenetically divergent classes of lanthipeptide synthetase to manipulate and exploit the dynamic lanthipeptide conformational ensemble, collectively enabling these biosynthetic enzymes to guide peptide maturation along specific trajectories to products with distinct macrocyclic topology and biological activity.
Assuntos
Ligases , Peptídeos , Sequência de Aminoácidos , Peptídeos/química , Ligases/química , Ligases/metabolismo , Processamento de Proteína Pós-Traducional , BiologiaRESUMO
We present a potential mechanism for emergence of catalytic activity that is essential for survival, from a non-catalytic protein fold. The type B dihydrofolate reductase (DfrB) family of enzymes were first identified in pathogenic bacteria because their dihydrofolate reductase activity is sufficient to provide trimethoprim (TMP) resistance. DfrB enzymes are described as poorly evolved as a result of their unusual structural and kinetic features. No characterized protein shares sequence homology with DfrB enzymes; how they evolved to emerge in the modern resistome is unknown. In this work, we identify DfrB homologues from a database of putative and uncharacterized proteins. These proteins include an SH3-like fold homologous to the DfrB enzymes, embedded in a variety of additional structural domains. By means of functional, structural and biophysical characterization, we demonstrate that these distant homologues and their extracted SH3-like fold can display dihydrofolate reductase activity and confer TMP resistance. We provide evidence of tetrameric assembly and catalytic mechanism analogous to that of DfrB enzymes. These results contribute, to our knowledge, the first insights into a potential evolutionary path taken by this SH3-like fold to emerge in the modern resistome following introduction of TMP. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.
Assuntos
Oxirredutases , Tetra-Hidrofolato Desidrogenase , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/metabolismo , Antibacterianos , Farmacorresistência BacterianaRESUMO
Pathogenic bacteria utilize specialized macromolecular secretion systems to transport virulence factors across membrane(s) and manipulate their infected host. To date, 11 secretion systems have been identified, including the type IX secretion system (T9SS) associated with human, avian and farmed-fish diseases. As a bacterial secretion system, the T9SS also facilitates gliding motility and the degradation of different macromolecules by the secretion of metabolic enzymes in nonpathogenic bacteria. PorX is a highly conserved protein that regulates the transcription of essential T9SS components and additionally mediates the function of T9SS via direct interaction with PorL, the rotary motor protein of the T9SS. PorX is also a member of a two-component system regulatory cascade, where it serves as the response regulator that relays a signal transduced from a conserved sensor histidine kinase, PorY, to a designated sigma factor. Here, the recombinant expression and purification of PorX homologous proteins from the pathogenic bacterium Porphyromonas gingivalis and the nonpathogenic bacterium Flavobacterium johnsoniae are reported. A bioinformatical characterization of the different domains comprising the PorX protein is also provided, and the crystallization and X-ray analysis of PorX from F. johnsoniae are reported.
Assuntos
Proteínas de Bactérias , Fator sigma , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/genética , Sistemas de Secreção Bacterianos/metabolismo , Cristalização , Cristalografia por Raios X , Histidina Quinase/metabolismo , Humanos , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/metabolismo , Fator sigma/metabolismo , Fatores de Virulência/metabolismoRESUMO
1-Aminocyclopropane-1-carboxylic acid (ACC) deaminase (ACCD) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that cleaves the cyclopropane ring of ACC, to give α-ketobutyric acid and ammonia as products. The cleavage of the C(α)-C(ß) bond of an amino acid substrate is a rare event in PLP-dependent enzyme catalysis. Potential chemical mechanisms involving nucleophile- or acid-catalyzed cyclopropane ring opening have been proposed for the unusual transformation catalyzed by ACCD, but the actual mode of cyclopropane ring cleavage remains obscure. In this report, we aim to elucidate the mechanistic features of ACCD catalysis by investigating the kinetic properties of ACCD from Pseudomonas sp. ACP and several of its mutant enzymes. Our studies suggest that the pK(a) of the conserved active site residue, Tyr294, is lowered by a hydrogen bonding interaction with a second conserved residue, Tyr268. This allows Tyr294 to deprotonate the incoming amino group of ACC to initiate the aldimine exchange reaction between ACC and the PLP coenzyme and also likely helps to activate Tyr294 for a role as a nucleophile to attack and cleave the cyclopropane ring of the substrate. In addition, solvent kinetic isotope effect (KIE), proton inventory, and (13)C KIE studies of the wild type enzyme suggest that the C(α)-C(ß) bond cleavage step in the chemical mechanism is at least partially rate-limiting under k(cat)/K(m) conditions and is likely preceded in the mechanism by a partially rate-limiting step involving the conversion of a stable gem-diamine intermediate into a reactive external aldimine intermediate that is poised for cyclopropane ring cleavage. When viewed within the context of previous mechanistic and structural studies of ACCD enzymes, our studies are most consistent with a mode of cyclopropane ring cleavage involving nucleophilic catalysis by Tyr294.