RESUMEN
Enzyme-based depolymerization is a viable approach for recycling of poly(ethylene terephthalate) (PET). PETase from Ideonella sakaiensis (IsPETase) is capable of PET hydrolysis under mild conditions but suffers from concentration-dependent inhibition. In this study, this inhibition is found to be dependent on incubation time, the solution conditions, and PET surface area. Furthermore, this inhibition is evident in other mesophilic PET-degrading enzymes to varying degrees, independent of the level of PET depolymerization activity. The inhibition has no clear structural basis, but moderately thermostable IsPETase variants exhibit reduced inhibition, and the property is completely absent in the highly thermostable HotPETase, previously engineered by directed evolution, which simulations suggest results from reduced flexibility around the active site. This work highlights a limitation in applying natural mesophilic hydrolases for PET hydrolysis and reveals an unexpected positive outcome of engineering these enzymes for enhanced thermostability.
Asunto(s)
Ácidos Ftálicos , Tereftalatos Polietilenos , Tereftalatos Polietilenos/química , Hidrolasas , Ácidos Ftálicos/química , EtilenosRESUMEN
Deciphering the mechanisms of bacterial fatty acid biosynthesis is crucial for both the engineering of bacterial hosts to produce fatty acid-derived molecules and the development of new antibiotics. However, gaps in our understanding of the initiation of fatty acid biosynthesis remain. Here, we demonstrate that the industrially relevant microbe Pseudomonas putida KT2440 contains three distinct pathways to initiate fatty acid biosynthesis. The first two routes employ conventional ß-ketoacyl-ACP synthase III enzymes, FabH1 and FabH2, that accept short- and medium-chain-length acyl-CoAs, respectively. The third route utilizes a malonyl-ACP decarboxylase enzyme, MadB. A combination of exhaustive in vivo alanine-scanning mutagenesis, in vitro biochemical characterization, X-ray crystallography, and computational modeling elucidate the presumptive mechanism of malonyl-ACP decarboxylation via MadB. Given that functional homologs of MadB are widespread throughout domain Bacteria, this ubiquitous alternative fatty acid initiation pathway provides new opportunities to target a range of biotechnology and biomedical applications.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa , Pseudomonas putida , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/genética , Mutagénesis , Ácidos GrasosRESUMEN
Lignin valorization is being intensely pursued via tandem catalytic depolymerization and biological funneling to produce single products. In many lignin depolymerization processes, aromatic dimers and oligomers linked by carbon-carbon bonds remain intact, necessitating the development of enzymes capable of cleaving these compounds to monomers. Recently, the catabolism of erythro-1,2-diguaiacylpropane-1,3-diol (erythro-DGPD), a ring-opened lignin-derived ß-1 dimer, was reported in Novosphingobium aromaticivorans. The first enzyme in this pathway, LdpA (formerly LsdE), is a member of the nuclear transport factor 2 (NTF-2)-like structural superfamily that converts erythro-DGPD to lignostilbene through a heretofore unknown mechanism. In this study, we performed biochemical, structural, and mechanistic characterization of the N. aromaticivorans LdpA and another homolog identified in Sphingobium sp. SYK-6, for which activity was confirmed in vivo. For both enzymes, we first demonstrated that formaldehyde is the C1 reaction product, and we further demonstrated that both enantiomers of erythro-DGPD were transformed simultaneously, suggesting that LdpA, while diastereomerically specific, lacks enantioselectivity. We also show that LdpA is subject to a severe competitive product inhibition by lignostilbene. Three-dimensional structures of LdpA were determined using X-ray crystallography, including substrate-bound complexes, revealing several residues that were shown to be catalytically essential. We used density functional theory to validate a proposed mechanism that proceeds via dehydroxylation and formation of a quinone methide intermediate that serves as an electron sink for the ensuing deformylation. Overall, this study expands the range of chemistry catalyzed by the NTF-2-like protein family to a prevalent lignin dimer through a cofactorless deformylation reaction.
Asunto(s)
Liasas , Lignina/metabolismo , Proteínas Bacterianas/metabolismo , Oxidorreductasas/metabolismo , EstereoisomerismoRESUMEN
Enzymatic deconstruction of poly(ethylene terephthalate) (PET) is under intense investigation, given the ability of hydrolase enzymes to depolymerize PET to its constituent monomers near the polymer glass transition temperature. To date, reported PET hydrolases have been sourced from a relatively narrow sequence space. Here, we identify additional PET-active biocatalysts from natural diversity by using bioinformatics and machine learning to mine 74 putative thermotolerant PET hydrolases. We successfully express, purify, and assay 51 enzymes from seven distinct phylogenetic groups; observing PET hydrolysis activity on amorphous PET film from 37 enzymes in reactions spanning pH from 4.5-9.0 and temperatures from 30-70 °C. We conduct PET hydrolysis time-course reactions with the best-performing enzymes, where we observe differences in substrate selectivity as function of PET morphology. We employed X-ray crystallography and AlphaFold to examine the enzyme architectures of all 74 candidates, revealing protein folds and accessory domains not previously associated with PET deconstruction. Overall, this study expands the number and diversity of thermotolerant scaffolds for enzymatic PET deconstruction.
Asunto(s)
Hidrolasas , Tereftalatos Polietilenos , Hidrolasas/metabolismo , Tereftalatos Polietilenos/química , Filogenia , Hidrólisis , EtilenosRESUMEN
The actinobacterium Rhodococcus jostii RHA1 grows on a remarkable variety of aromatic compounds and has been studied for applications ranging from the degradation of polychlorinated biphenyls to the valorization of lignin, an underutilized component of biomass. In RHA1, the catabolism of two classes of lignin-derived compounds, alkylphenols and alkylguaiacols, involves a phylogenetically distinct extradiol dioxygenase, AphC, previously misannotated as BphC, an enzyme involved in biphenyl catabolism. To better understand the role of AphC in RHA1 catabolism, we first showed that purified AphC had highest apparent specificity for 4-propylcatechol (kcat/KM â¼106 M-1 s-1), and its apparent specificity for 4-alkylated substrates followed the trend for alkylguaiacols: propyl > ethyl > methyl > phenyl > unsubstituted. We also show AphC only poorly cleaved 3-phenylcatechol, the preferred substrate of BphC. Moreover, AphC and BphC cleaved 3-phenylcatechol and 4-phenylcatechol with different regiospecificities, likely due to the substrates' binding mode. A crystallographic structure of the AphC·4-ethylcatechol binary complex to 1.59 Å resolution revealed that the catechol is bound to the active site iron in a bidentate manner and that the substrate's alkyl side chain is accommodated by a hydrophobic pocket. Finally, we show RHA1 grows on a mixture of 4-ethylguaiacol and guaiacol, simultaneously catabolizing these substrates through meta-cleavage and ortho-cleavage pathways, respectively, suggesting that the specificity of AphC helps to prevent the routing of catechol through the Aph pathway. Overall, this study contributes to our understanding of the bacterial catabolism of aromatic compounds derived from lignin, and the determinants of specificity in extradiol dioxygenases.
Asunto(s)
Dioxigenasas , Rhodococcus , Catecoles , Dioxigenasas/metabolismo , Hidrolasas/metabolismo , Lignina/metabolismo , Oxigenasas/metabolismoRESUMEN
SignificanceMore than 400 million tons of plastic waste is produced each year, the overwhelming majority of which ends up in landfills. Bioconversion strategies aimed at plastics have emerged as important components of enabling a circular economy for synthetic plastics, especially those that exhibit chemically similar linkages to those found in nature, such as polyesters. The enzyme system described in this work is essential for mineralization of the xenobiotic components of poly(ethylene terephthalate) (PET) in the biosphere. Our description of its structure and substrate preferences lays the groundwork for in vivo or ex vivo engineering of this system for PET upcycling.
Asunto(s)
Dioxigenasas , Ácidos Ftálicos , Plásticos/química , Tereftalatos Polietilenos/químicaRESUMEN
The transformation of 4-hydroxybenzoate (4-HBA) to protocatechuate (PCA) is catalyzed by flavoprotein oxygenases known as para-hydroxybenzoate-3-hydroxylases (PHBHs). In Pseudomonas putida KT2440 (P. putida) strains engineered to convert lignin-related aromatic compounds to muconic acid (MA), PHBH activity is rate-limiting, as indicated by the accumulation of 4-HBA, which ultimately limits MA productivity. Here, we hypothesized that replacement of PobA, the native P. putida PHBH, with PraI, a PHBH from Paenibacillus sp. JJ-1b with a broader nicotinamide cofactor preference, could alleviate this bottleneck. Biochemical assays confirmed the strict preference of NADPH for PobA, while PraI can utilize either NADH or NADPH. Kinetic assays demonstrated that both PobA and PraI can utilize NADPH with comparable catalytic efficiency and that PraI also efficiently utilizes NADH at roughly half the catalytic efficiency. The X-ray crystal structure of PraI was solved and revealed absolute conservation of the active site architecture to other PHBH structures despite their differing cofactor preferences. To understand the effect in vivo, we compared three P. putida strains engineered to produce MA from p-coumarate (pCA), showing that expression of praI leads to lower 4-HBA accumulation and decreased NADP+/NADPH ratios relative to strains harboring pobA, indicative of a relieved 4-HBA bottleneck due to increased NADPH availability. In bioreactor cultivations, a strain exclusively expressing praI achieved a titer of 40 g/L MA at 100% molar yield and a productivity of 0.5 g/L/h. Overall, this study demonstrates the benefit of sampling readily available natural enzyme diversity for debottlenecking metabolic flux in an engineered strain for microbial conversion of lignin-derived compounds to value-added products.
Asunto(s)
Pseudomonas putida , Hidroxibenzoatos/metabolismo , Hidroxilación , Parabenos , Pseudomonas putida/genética , Pseudomonas putida/metabolismoRESUMEN
Actinobacterial 2-hydroxyacyl-CoA lyase reversibly catalyzes the thiamine diphosphate-dependent cleavage of 2-hydroxyisobutyryl-CoA to formyl-CoA and acetone. This enzyme has great potential for use in synthetic one-carbon assimilation pathways for sustainable production of chemicals, but lacks details of substrate binding and reaction mechanism for biochemical reengineering. We determined crystal structures of the tetrameric enzyme in the closed conformation with bound substrate, covalent postcleavage intermediate, and products, shedding light on active site architecture and substrate interactions. Together with molecular dynamics simulations of the covalent precleavage complex, the complete catalytic cycle is structurally portrayed, revealing a proton transfer from the substrate acyl Cß hydroxyl to residue E493 that returns it subsequently to the postcleavage Cα-carbanion intermediate. Kinetic parameters obtained for mutants E493A, E493Q, and E493K confirm the catalytic role of E493 in the WT enzyme. However, the 10- and 50-fold reduction in lyase activity in the E493A and E493Q mutants, respectively, compared with WT suggests that water molecules may contribute to proton transfer. The putative catalytic glutamate is located on a short α-helix close to the active site. This structural feature appears to be conserved in related lyases, such as human 2-hydroxyacyl-CoA lyase 2. Interestingly, a unique feature of the actinobacterial 2-hydroxyacyl-CoA lyase is a large C-terminal lid domain that, together with active site residues L127 and I492, restricts substrate size to ≤C5 2-hydroxyacyl residues. These details about the catalytic mechanism and determinants of substrate specificity pave the ground for designing tailored catalysts for acyloin condensations for one-carbon and short-chain substrates in biotechnological applications.
Asunto(s)
Acilcoenzima A , Liasas , Acilcoenzima A/química , Acilcoenzima A/metabolismo , Carbono , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Liasas/química , Liasas/metabolismo , Protones , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Invited for this month's cover is the BOTTLE Consortium, featuring Gregg Beckham's laboratory from NREL and John McGeehan's laboratory from the University of Portsmouth. The cover image shows the application of poly(ethylene terephthalate) (PET) hydrolase enzymes on post-consumer waste plastic, towards the development of an enzymatic PET recycling strategy. The Full Paper itself is available at 10.1002/cssc.202101932.
Asunto(s)
Burkholderiales , Hidrolasas , Plásticos , Tereftalatos Polietilenos , ReciclajeRESUMEN
There is keen interest to develop new technologies to recycle the plastic poly(ethylene terephthalate) (PET). To this end, the use of PET-hydrolyzing enzymes has shown promise for PET deconstruction to its monomers, terephthalate (TPA) and ethylene glycol (EG). Here, the Ideonella sakaiensis PETase wild-type enzyme was compared to a previously reported improved variant (W159H/S238F). The thermostability of each enzyme was compared and a 1.45â Å resolution structure of the mutant was described, highlighting changes in the substrate binding cleft compared to the wild-type enzyme. Subsequently, the performance of the wild-type and variant enzyme was compared as a function of temperature, substrate morphology, and reaction mixture composition. These studies showed that reaction temperature had the strongest influence on performance between the two enzymes. It was also shown that both enzymes achieved higher levels of PET conversion for substrates with moderate crystallinity relative to amorphous substrates. Finally, the impact of product accumulation on reaction progress was assessed for the hydrolysis of both PET and bis(2-hydroxyethyl) terephthalate (BHET). Each enzyme displayed different inhibition profiles to mono(2-hydroxyethyl) terephthalate (MHET) and TPA, while both were sensitive to inhibition by EG. Overall, this study highlights the importance of reaction conditions, substrate selection, and product accumulation for catalytic performance of PET-hydrolyzing enzymes, which have implications for enzyme screening in the development of enzyme-based polyester recycling.
Asunto(s)
Hidrolasas , Tereftalatos Polietilenos , Hidrólisis , Plásticos , ReciclajeRESUMEN
This study describes a novel series of UDP-N-acetylglucosamine acyltransferase (LpxA) inhibitors that was identified through affinity-mediated selection from a DNA-encoded compound library. The original hit was a selective inhibitor of Pseudomonas aeruginosa LpxA with no activity against Escherichia coli LpxA. The biochemical potency of the series was optimized through an X-ray crystallography-supported medicinal chemistry program, resulting in compounds with nanomolar activity against P. aeruginosa LpxA (best half-maximal inhibitory concentration (IC50) <5 nM) and cellular activity against P. aeruginosa (best minimal inhibitory concentration (MIC) of 4 µg/mL). Lack of activity against E. coli was maintained (IC50 > 20 µM and MIC > 128 µg/mL). The mode of action of analogues was confirmed through genetic analyses. As expected, compounds were active against multidrug-resistant isolates. Further optimization of pharmacokinetics is needed before efficacy studies in mouse infection models can be attempted. To our knowledge, this is the first reported LpxA inhibitor series with selective activity against P. aeruginosa.
Asunto(s)
Aciltransferasas/antagonistas & inhibidores , Antibacterianos/farmacología , Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Antibacterianos/química , Cristalografía por Rayos X , Farmacorresistencia Bacteriana/efectos de los fármacos , Inhibidores Enzimáticos/química , Escherichia coli/enzimología , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Relación Estructura-ActividadRESUMEN
In Bacteroidetes, one of the dominant phyla of the mammalian gut, active uptake of large nutrients across the outer membrane is mediated by SusCD protein complexes via a "pedal bin" transport mechanism. However, many features of SusCD function in glycan uptake remain unclear, including ligand binding, the role of the SusD lid and the size limit for substrate transport. Here we characterise the ß2,6 fructo-oligosaccharide (FOS) importing SusCD from Bacteroides thetaiotaomicron (Bt1762-Bt1763) to shed light on SusCD function. Co-crystal structures reveal residues involved in glycan recognition and suggest that the large binding cavity can accommodate several substrate molecules, each up to ~2.5 kDa in size, a finding supported by native mass spectrometry and isothermal titration calorimetry. Mutational studies in vivo provide functional insights into the key structural features of the SusCD apparatus and cryo-EM of the intact dimeric SusCD complex reveals several distinct states of the transporter, directly visualising the dynamics of the pedal bin transport mechanism.
Asunto(s)
Proteínas Bacterianas/metabolismo , Microbioma Gastrointestinal , Polisacáridos/metabolismo , Simbiosis , Proteínas Bacterianas/química , Microscopía por Crioelectrón , Ligandos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Oligosacáridos/química , Polisacáridos/química , Conformación Proteica , Relación Estructura-ActividadRESUMEN
An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
There is an urgent need for new antibiotics against Gram-negative pathogens that are resistant to carbapenem and third-generation cephalosporins, against which antibiotics of last resort have lost most of their efficacy. Here we describe a class of synthetic antibiotics inspired by scaffolds derived from natural products. These chimeric antibiotics contain a ß-hairpin peptide macrocycle linked to the macrocycle found in the polymyxin and colistin family of natural products. They are bactericidal and have a mechanism of action that involves binding to both lipopolysaccharide and the main component (BamA) of the ß-barrel folding complex (BAM) that is required for the folding and insertion of ß-barrel proteins into the outer membrane of Gram-negative bacteria. Extensively optimized derivatives show potent activity against multidrug-resistant pathogens, including all of the Gram-negative members of the ESKAPE pathogens1. These derivatives also show favourable drug properties and overcome colistin resistance, both in vitro and in vivo. The lead candidate is currently in preclinical toxicology studies that-if successful-will allow progress into clinical studies that have the potential to address life-threatening infections by the Gram-negative pathogens, and thus to resolve a considerable unmet medical need.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Farmacorresistencia Microbiana , Bacterias Gramnegativas/efectos de los fármacos , Peptidomiméticos/química , Peptidomiméticos/farmacología , Animales , Antibacterianos/efectos adversos , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Productos Biológicos/química , Descubrimiento de Drogas , Farmacorresistencia Microbiana/efectos de los fármacos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Fluorescencia , Bacterias Gramnegativas/genética , Bacterias Gramnegativas/patogenicidad , Humanos , Lipopolisacáridos/química , Compuestos Macrocíclicos/efectos adversos , Compuestos Macrocíclicos/química , Compuestos Macrocíclicos/farmacología , Masculino , Ratones , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Microscopía Electrónica de Transmisión , Modelos Moleculares , Mutación , Peptidomiméticos/efectos adversos , Etiquetas de FotoafinidadRESUMEN
2-Hydroxyisobutyric acid (2-HIBA) is a biomarker of adiposity and associated metabolic diseases such as diabetes mellitus. It is also formed in the bacterial degradation pathway of the fuel oxygenate methyl tert-butyl ether (MTBE), requiring thioesterification with CoA prior to isomerization to 3-hydroxybutyryl-CoA by B12-dependent acyl-CoA mutases. Here, we identify the adenylating enzymes superfamily member 2-HIBA-CoA ligase (HCL) in the MTBE-degrading bacterium Aquincola tertiaricarbonis L108 by knockout experiments. To characterize this central enzyme of 2-HIBA metabolism, ligase activity kinetics of purified HCL and its X-ray crystal structures were studied. We analyzed the enzyme in three states, which differ in the orientation of the two enzyme domains. A 154° rotation of the C-terminal domain accompanies the switch from the adenylate- into the thioester-forming state. Furthermore, a third conformation was obtained, which differs by 50° and 130° from the adenylation and thioesterification states, respectively. Phylogenetic and structural analysis reveals that HCL defines a new subgroup within phenylacetate-CoA ligases (PCLs) thus far described to exclusively accept aromatic acyl substrates. In contrast, kinetic characterization clearly demonstrated that HCL catalyzes CoA activation of several aliphatic short-chain carboxylic acids, preferentially 2-HIBA. Compared to the classical PCL representatives PaaK1 and PaaK2 of Burkholderia cenocepacia J2315, the acyl binding pocket of HCL is significantly smaller and more polar, due to unique active-site residues Y164 and S239 forming H-bonds with the OH-group of the acyl substrate moiety. Furthermore, HCL and PaaK topologies illustrate the evolutionary steps leading from a homodimeric to the fused monomeric core fold found in other ligases.
Asunto(s)
Proteínas Bacterianas/química , Burkholderiales/química , Coenzima A Ligasas/química , Proteínas Bacterianas/metabolismo , Burkholderiales/metabolismo , Dominio Catalítico , Coenzima A Ligasas/metabolismo , Cristalografía por Rayos X , Hidroxibutiratos/metabolismo , Modelos Moleculares , Conformación Proteica , Especificidad por SustratoRESUMEN
The insertase BamA is an essential protein of the bacterial outer membrane. Its 16-stranded transmembrane ß-barrel contains a lateral gate as a key functional element. This gate is formed by the C-terminal half of the last ß-strand. The BamA barrel was previously found to sample different conformations in aqueous solution, as well as different gate-open, gate-closed, and collapsed conformations in X-ray crystallography and cryo-electron microscopy structures. Here, we report the successful identification of conformation-selective nanobodies that stabilize BamA in specific conformations. While the initial candidate generation and selection protocol was based on established alpaca immunization and phage display selection procedures, the final selection of nanobodies was enhanced by a solution NMR-based screening step to shortlist the targets for crystallization. In this way, three crystal structures of BamA-nanobody complexes were efficiently obtained, showing two types of nanobodies that indeed stabilized BamA in two different conformations, i.e., with open and closed lateral gate, respectively. Then, by correlating the structural data with high resolution NMR spectra, we could for the first time assign the BamA conformational solution ensemble to defined structural states. The new nanobodies will be valuable tools towards understanding the client insertion mechanism of BamA and towards developing improved antibiotics.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Modelos Moleculares , Conformación Proteica , Anticuerpos de Dominio Único/química , Proteínas de la Membrana Bacteriana Externa/antagonistas & inhibidores , Evaluación Preclínica de Medicamentos , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Anticuerpos de Dominio Único/farmacología , SolucionesRESUMEN
Research efforts to discover potential new antibiotics for Gram-negative bacteria suffer from high attrition rates due to the synergistic action of efflux systems and the limited permeability of the outer membrane (OM). One strategy to overcome the OM permeability barrier is to identify small molecules that are natural substrates for abundant OM channels and use such compounds as scaffolds for the design of efficiently permeating antibacterials. Here we present a multidisciplinary approach to identify such potential small-molecule scaffolds. Focusing on the pathogenic bacterium Acinetobacter baumannii, we use OM proteomics to identify DcaP as the most abundant channel during infection in rodents. The X-ray crystal structure of DcaP reveals a trimeric, porin-like structure and suggests that dicarboxylic acids are potential transport substrates. Electrophysiological experiments and all-atom molecular dynamics simulations confirm this notion and provide atomistic information on likely permeation pathways and energy barriers for several small molecules, including a clinically relevant ß-lactamase inhibitor.
Asunto(s)
Infecciones por Acinetobacter/metabolismo , Acinetobacter baumannii/metabolismo , Porinas/química , Porinas/metabolismo , Sulbactam/farmacología , Inhibidores de beta-Lactamasas/farmacología , Acinetobacter baumannii/efectos de los fármacos , Animales , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Cristalografía por Rayos X , Ácidos Dicarboxílicos/metabolismo , Ratones , Modelos Moleculares , Simulación de Dinámica Molecular , Conformación Proteica , Multimerización de Proteína , Proteómica , RatasRESUMEN
Acinetobacter baumannii is becoming a major threat to human health due to its multidrug resistance. This is owing in a large part to the low permeability of its outer membrane (OM), which prevents high internal antibiotic concentrations and makes antibiotic-resistance mechanisms more effective. To exploit OM channels as potential delivery vehicles for future antibiotics, structural information is required. One abundant OM protein in A. baumannii is Omp33. This protein has been reported to be important for the in vivo fitness and virulence of A. baumannii, but its structure is not known. Here, the X-ray crystal structure of Omp33 is reported at a resolution of 2.1â Å. Omp33 has a 14-ß-stranded barrel without stable extracellular loop constrictions. Instead, an extended and unusual periplasmic turn connecting ß-strands 2 and 3 is present, which folds into the pore lumen and completely blocks the aqueous channel. The Omp33 structure helps in understanding how A. baumannii OM proteins contribute to the low permeability of the cell envelope of this bacterium and suggests that Omp33 might function as a gated channel.
Asunto(s)
Acinetobacter baumannii/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Cristalografía por Rayos X , Acinetobacter baumannii/química , Secuencia de Aminoácidos , Humanos , Activación del Canal Iónico , Canales Iónicos/química , Modelos Moleculares , Estructura Terciaria de ProteínaRESUMEN
ß-barrel outer membrane proteins (Omps) are key functional components of the outer membranes of Gram-negative bacteria, mitochondria, and plastids. In bacteria, their biogenesis requires the ß-barrel-assembly machinery (Bam) with the central insertase BamA, but the exact translocation and insertion mechanism remains elusive. The BamA insertase features a loosely closed gating region between the first and last ß-strand 16. Here, we describe â¼70% complete sequence-specific NMR resonance assignments of the transmembrane region of the BamA ß-barrel in detergent micelles. On the basis of the assignments, NMR spectra show that the BamA barrel populates a conformational ensemble in slow exchange equilibrium, both in detergent micelles and lipid bilayer nanodiscs. Individual conformers can be selected from the ensemble by the introduction of a C-terminal strand extension, single-point mutations, or specific disulfide cross-linkings, and these modifications at the barrel seam are found to be allosterically coupled to sites at the entire barrel circumference. The resonance assignment provides a platform for mechanistic studies of BamA at atomic resolution, as well as for investigating interactions with potential antibiotic drugs and partner proteins.
RESUMEN
Small, hydrophilic molecules, including most important antibiotics in clinical use, cross the Gram-negative outer membrane through the water-filled channels provided by porins. We have determined the X-ray crystal structures of the principal general porins from three species of Enterobacteriaceae, namely Enterobacter aerogenes, Enterobacter cloacae, and Klebsiella pneumoniae, and determined their antibiotic permeabilities as well as those of the orthologues from Escherichia coli. Starting from the structure of the porins and molecules, we propose a physical mechanism underlying transport and condense it in a computationally efficient scoring function. The scoring function shows good agreement with in vitro penetration data and will enable the screening of virtual databases to identify molecules with optimal permeability through porins and help to guide the optimization of antibiotics with poor permeation.