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INTRODUCTION: Nuclear magnetic resonance (NMR) is widely used in metabolomics but it focusses on 1H over other NMR-active nuclei. OBJECTIVES: To evaluate the potential of alternative NMR-sensitive nuclei to generate useful metabolomic data. METHOD: Proton, carbon, phosphorus, and nitrogen-based NMR metabolomics was undertaken on extracts from mint and European honey bee tissue. RESULTS: Carbon NMR provided useful information but required larger sample sizes. Phosphorus produced overlapping peaks in one dimensional (1D) analysis but showed potential in 2D experiments. 15N NMR was found to not be sensitive enough for general metabolomic work. CONCLUSIONS: Alternative NMR active nuclei are useful for metabolomics.
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Imageamento por Ressonância Magnética , Metabolômica , Abelhas , Animais , Metabolômica/métodos , Espectroscopia de Ressonância Magnética/métodos , CarbonoRESUMO
Patients with indolent lymphoma undertaking recurrent or continuous B cell suppression are at risk of severe COVID-19. Patients and healthy controls (HC; N = 13) received two doses of BNT162b2: follicular lymphoma (FL; N = 35) who were treatment naïve (TN; N = 11) or received immunochemotherapy (ICT; N = 23) and Waldenström's macroglobulinemia (WM; N = 37) including TN (N = 9), ICT (N = 14), or treated with Bruton's tyrosine kinase inhibitors (BTKi; N = 12). Anti-spike immunoglobulin G (IgG) was determined by a high-sensitivity flow-cytometric assay, in addition to live-virus neutralization. Antigen-specific T cells were identified by coexpression of CD69/CD137 and CD25/CD134 on T cells. A subgroup (N = 29) were assessed for third mRNA vaccine response, including omicron neutralization. One month after second BNT162b2, median anti-spike IgG mean fluorescence intensity (MFI) in FL ICT patients (9977) was 25-fold lower than TN (245 898) and HC (228 255, p = .0002 for both). Anti-spike IgG correlated with lymphocyte count (r = .63; p = .002), and time from treatment (r = .56; p = .007), on univariate analysis, but only with lymphocyte count on multivariate analysis (p = .03). In the WM cohort, median anti-spike IgG MFI in BTKi patients (39 039) was reduced compared to TN (220 645, p = .0008) and HC (p < .0001). Anti-spike IgG correlated with neutralization of the delta variant (r = .62, p < .0001). Median neutralization titer for WM BTKi (0) was lower than HC (40, p < .0001) for early-clade and delta. All cohorts had functional T cell responses. Median anti-spike IgG decreased 4-fold from second to third dose (p = .004). Only 5 of 29 poor initial responders assessed after third vaccination demonstrated seroconversion and improvement in neutralization activity, including to the omicron variant.
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COVID-19 , Linfoma não Hodgkin , Humanos , Imunoglobulina G , SARS-CoV-2 , Vacina BNT162 , COVID-19/prevenção & controle , Linfócitos T , Anticorpos Antivirais , Anticorpos Neutralizantes , VacinaçãoRESUMO
To engineer Mo-dependent nitrogenase function in plants, expression of the structural proteins NifD and NifK will be an absolute requirement. Although mitochondria have been established as a suitable eukaryotic environment for biosynthesis of oxygen-sensitive enzymes such as NifH, expression of NifD in this organelle has proven difficult due to cryptic NifD degradation. Here, we describe a solution to this problem. Using molecular and proteomic methods, we found NifD degradation to be a consequence of mitochondrial endoprotease activity at a specific motif within NifD. Focusing on this functionally sensitive region, we designed NifD variants comprising between one and three amino acid substitutions and distinguished several that were resistant to degradation when expressed in both plant and yeast mitochondria. Nitrogenase activity assays of these resistant variants in Escherichia coli identified a subset that retained function, including a single amino acid variant (Y100Q). We found that other naturally occurring NifD proteins containing alternate amino acids at the Y100 position were also less susceptible to degradation. The Y100Q variant also enabled expression of a NifD(Y100Q)-linker-NifK translational polyprotein in plant mitochondria, confirmed by identification of the polyprotein in the soluble fraction of plant extracts. The NifD(Y100Q)-linker-NifK retained function in bacterial nitrogenase assays, demonstrating that this polyprotein permits expression of NifD and NifK in a defined stoichiometry supportive of activity. Our results exemplify how protein design can overcome impediments encountered when expressing synthetic proteins in novel environments. Specifically, these findings outline our progress toward the assembly of the catalytic unit of nitrogenase within mitochondria.
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Genes Bacterianos/genética , Mitocôndrias/genética , Mitocôndrias/fisiologia , Proteínas de Plantas/genética , Plantas/genética , Substituição de Aminoácidos/genética , Escherichia coli/genética , Fixação de Nitrogênio/genética , Nitrogenase/genética , Poliproteínas/genética , Proteômica/instrumentaçãoRESUMO
A lack of toxicity data quantifying responses of Australian native mammals to agricultural pesticides prompted an investigation into the sensitivity of the stripe-faced dunnart, Sminthopsis macroura (Gould 1845) to the insecticide, fipronil (5-amino-3-cyano-1-(2,6-dichloro-4-trifluoromethylphenyl)-4-trifluoromethylsulfinyl pyrazole, CAS No. 120068-37-3). Using the Up-And-Down method for determining acute oral toxicity in mammals (OECD) median lethal dose estimates of 990 mg kg-1 (95% confidence interval (CI) = 580.7-4770.0 mg kg-1) and 270.4 mg kg-1 (95% CI = 0.0->20,000.0 mg kg-1) were resolved for male and female S. macroura, respectively. The difference between median lethal dose estimates for males and females may have been influenced by the older ages of two female dunnarts. Consequently, further modelling of female responses to fipronil doses used the following assumptions: (a) death at 2000 mg kg-1, (b) survival at 500 mg kg-1 and (c) a differential response (both survival and death) at 990 mg kg-1. This modelling revealed median lethal dose estimates for female S. macroura of 669.1 mg kg-1 (95% CI = 550-990 mg kg-1; assuming death at 990 mg kg-1) and 990 mg kg-1 (95% CI = 544.7-1470 mg kg-1; assuming survival at 990 mg kg-1). These median lethal dose estimates are 3-10-fold higher than available LD50 values of 94 mg kg-1 for a similarly sized eutherian mammal, Mus musculus (L. 1758) and 97 mg kg-1 for Rattus norvegicus (Birkenhout 1769). Implications for pesticide risk assessments in Australia are discussed.
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Inseticidas , Marsupiais , Praguicidas , Animais , Austrália , Feminino , Inseticidas/toxicidade , Masculino , Marsupiais/fisiologia , Camundongos , Pirazóis/toxicidade , Ratos , Medição de RiscoRESUMO
The ability to produce large numbers of pesticide-exposed insects (e.g. crickets) is important for feeding studies into the effects of pesticides on key predatory species. House crickets (Acheta domesticus L. 1758) were submersed in serial dilutions of the pesticides, fenitrothion and fipronil, used for the control of locusts in Australia, and then rapidly frozen for residue analysis. Good correlations were found between increasing concentrations of serial pesticide dilutions and the resultant residual concentrations of the parent compounds in crickets, with R2 values of 0.949 (fenitrothion) and 0.946 (fipronil). R2 values for the much less abundant fipronil metabolites were lower 0.858 (sulfone), 0.368 (desulfinyl) and 0.785 (sulfide). This method enables insecticide exposure mimicking the field conditions to be assessed, and can be done immediately prior to an experiment. This ensures locusts remain alive when introduced to the feeding chambers, and enables multiple prey items to be dosed with a known pesticide burden.
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Inseticidas , Praguicidas , Animais , Austrália , Fenitrotion/toxicidade , Inseticidas/análise , Inseticidas/toxicidade , Invertebrados , Praguicidas/toxicidadeRESUMO
Pinoresinol is a dimer of two ß-ß'-linked coniferyl alcohol molecules. It is both a plant defense molecule synthesized through the shikimic acid pathway and a representative of several ß-ß-linked dimers produced during the microbial degradation of lignin in dead plant material. Until now, little has been known about the bacterial catabolism of such dimers. Here we report the isolation of the efficient (+)-pinoresinol-mineralizing Pseudomonas sp. strain SG-MS2 and its catabolic pathway. Degradation of pinoresinol in this strain is inducible and proceeds via a novel oxidative route, which is in contrast to the previously reported reductive transformation by other bacteria. Based on enzyme assays and bacterial growth, cell suspension, and resting cell studies, we provide conclusive evidence that pinoresinol degradation in strain SG-MS2 is initiated by benzylic hydroxylation, generating a hemiketal via a quinone methide intermediate, which is then hydrated at the benzylic carbon by water. The hemiketal, which stays in equilibrium with the corresponding keto alcohol, undergoes an aryl-alkyl cleavage to generate a lactone and 2-methoxyhydroquinone. While the fate of 2-methoxyhydroquinone is not investigated further, it is assumed to be assimilated by ring cleavage. The lactone is further metabolized via two routes, namely, lactone ring cleavage and benzylic hydroxylation via a quinone methide intermediate, as described above. The resulting hemiketal again exists in equilibrium with a keto alcohol. Our evidence suggests that both routes of lactone metabolism lead to vanillin and vanillic acid, which we show can then be mineralized by strain SG-MS2.IMPORTANCE The oxidative catabolism of (+)-pinoresinol degradation elucidated here is fundamentally different from the reductive cometabolism reported for two previously characterized bacteria. Our findings open up new opportunities to use lignin for the biosynthesis of vanillin, a key flavoring agent in foods, beverages, and pharmaceuticals, as well as various new lactones. Our work also has implications for the study of new pinoresinol metabolites in human health. The enterodiol and enterolactone produced through reductive transformation of pinoresinol by gut microbes have already been associated with decreased risks of cancer and cardiovascular diseases. The metabolites from oxidative metabolism we find here also deserve attention in this respect.
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Calcificação Fisiológica/fisiologia , Furanos/metabolismo , Lignanas/metabolismo , Redes e Vias Metabólicas , Pseudomonas/isolamento & purificação , Pseudomonas/metabolismo , 4-Butirolactona/análogos & derivados , 4-Butirolactona/metabolismo , Benzaldeídos/metabolismo , Microbioma Gastrointestinal/fisiologia , Humanos , Lignina/metabolismo , Minerais/metabolismo , Pseudomonas/genéticaRESUMO
A defining feature of mycobacterial redox metabolism is the use of an unusual deazaflavin cofactor, F420 This cofactor enhances the persistence of environmental and pathogenic mycobacteria, including after antimicrobial treatment, although the molecular basis for this remains to be understood. In this work, we explored our hypothesis that F420 enhances persistence by serving as a cofactor in antimicrobial-detoxifying enzymes. To test this, we performed a series of phenotypic, biochemical, and analytical chemistry studies in relation to the model soil bacterium Mycobacterium smegmatis Mutant strains unable to synthesize or reduce F420 were found to be more susceptible to a wide range of antibiotic and xenobiotic compounds. Compounds from three classes of antimicrobial compounds traditionally resisted by mycobacteria inhibited the growth of F420 mutant strains at subnanomolar concentrations, namely, furanocoumarins (e.g., methoxsalen), arylmethanes (e.g., malachite green), and quinone analogues (e.g., menadione). We demonstrated that promiscuous F420H2-dependent reductases directly reduce these compounds by a mechanism consistent with hydride transfer. Moreover, M. smegmatis strains unable to make F420H2 lost the capacity to reduce and detoxify representatives of the furanocoumarin and arylmethane compound classes in whole-cell assays. In contrast, mutant strains were only slightly more susceptible to clinical antimycobacterials, and this appeared to be due to indirect effects of F420 loss of function (e.g., redox imbalance) rather than loss of a detoxification system. Together, these data show that F420 enhances antimicrobial resistance in mycobacteria and suggest that one function of the F420H2-dependent reductases is to broaden the range of natural products that mycobacteria and possibly other environmental actinobacteria can reductively detoxify.IMPORTANCE This study reveals that a unique microbial cofactor, F420, is critical for antimicrobial resistance in the environmental actinobacterium Mycobacterium smegmatis We show that a superfamily of redox enzymes, the F420H2-dependent reductases, can reduce diverse antimicrobials in vitro and in vivoM. smegmatis strains unable to make or reduce F420 become sensitive to inhibition by these antimicrobial compounds. This suggests that mycobacteria have harnessed the unique properties of F420 to reduce structurally diverse antimicrobials as part of the antibiotic arms race. The F420H2-dependent reductases that facilitate this process represent a new class of antimicrobial-detoxifying enzymes with potential applications in bioremediation and biocatalysis.
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Medidas de Resultados Relatados pelo Paciente , Sistema de Registros , Macroglobulinemia de Waldenstrom/epidemiologia , Antineoplásicos/uso terapêutico , Austrália , Ensaios Clínicos como Assunto , Estudos de Viabilidade , Saúde Global/estatística & dados numéricos , Humanos , Padrões de Prática Médica , Qualidade de Vida , Macroglobulinemia de Waldenstrom/tratamento farmacológico , Macroglobulinemia de Waldenstrom/psicologiaRESUMO
Lignin is a complex aromatic polymer found in plant cell walls that makes up 15 to 40% of plant biomass. The degradation of lignin substructures by bacteria is of emerging interest because it could provide renewable alternative feedstocks and intermediates for chemical manufacturing industries. We have isolated a bacterium, strain SG61-1L, that rapidly degrades all of the stereoisomers of one lignin substructure, guaiacylglycerol-ß-guaiacyl ether (GGE), which contains a key ß-O-4 linkage found in most intermonomer linkages in lignin. In an effort to understand the rapid degradation of GGE by this bacterium, we heterologously expressed and kinetically characterized a suite of dehydrogenase candidates for the first known step of GGE degradation. We identified a clade of active GGE dehydrogenases and also several other dehydrogenases outside this clade that were all able to oxidize GGE. Several candidates exhibited stereoselectivity toward the GGE stereoisomers, while others had higher levels of catalytic performance than previously described GGE dehydrogenases for all four stereoisomers, indicating a variety of potential applications for these enzymes in the manufacture of lignin-derived commodities.
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Proteínas de Bactérias/genética , Guaifenesina/análogos & derivados , Lignina/metabolismo , Sphingomonadaceae/genética , Desidrogenase do Álcool de Açúcar/genética , Proteínas de Bactérias/metabolismo , Catálise , Guaifenesina/metabolismo , Cinética , Oxirredução , Filogenia , Sphingomonadaceae/metabolismo , Estereoisomerismo , Desidrogenase do Álcool de Açúcar/metabolismoRESUMO
The activity of the allophanate hydrolase from Pseudomonas sp. strain ADP, AtzF, provides the final hydrolytic step for the mineralization of s-triazines, such as atrazine and cyanuric acid. Indeed, the action of AtzF provides metabolic access to two of the three nitrogens in each triazine ring. The X-ray structure of the N-terminal amidase domain of AtzF reveals that it is highly homologous to allophanate hydrolases involved in a different catabolic process in other organisms (i.e., the mineralization of urea). The smaller C-terminal domain does not appear to have a physiologically relevant catalytic function, as reported for the allophanate hydrolase of Kluyveromyces lactis, when purified enzyme was tested in vitro. However, the C-terminal domain does have a function in coordinating the quaternary structure of AtzF. Interestingly, we also show that AtzF forms a large, ca. 660-kDa, multienzyme complex with AtzD and AtzE that is capable of mineralizing cyanuric acid. The function of this complex may be to channel substrates from one active site to the next, effectively protecting unstable metabolites, such as allophanate, from solvent-mediated decarboxylation to a dead-end metabolic product.
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Alofanato Hidrolase/química , Alofanato Hidrolase/metabolismo , Amidoidrolases/química , Amidoidrolases/metabolismo , Triazinas/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Conformação Proteica , Pseudomonas/enzimologiaRESUMO
INTRODUCTION: Many types of prostate cancer present minimal risk to a man's lifespan or well-being, but existing terminology makes it difficult for men to distinguish these from high-risk prostate cancers. This study aims to explore whether using an alternative label for low-risk prostate cancer influences management choice and anxiety levels among Australian men and their partners. METHODS AND ANALYSIS: We will run two separate studies for Australian men and Australian women with a male partner. Both studies are between-subjects factorial (3×2) randomised online hypothetical experiments. Following consent, eligible participants will be randomised 1:1:1 to three labels: 'low-risk prostate cancer, Gleason Group 1', 'low-risk prostate neoplasm' or 'low-risk prostate lesion'. Participants will then undergo a second randomisation step with 1:1 allocation to the provision of detailed information on the benefits and harms of different management choices versus the provision of less detailed information about management choices. The required sample sizes are 1290 men and 1410 women. The primary outcome is the participant choice of their preferred management strategy: no immediate treatment (prostate-specific antigen (PSA)-based monitoring or active surveillance using PSA, MRI, biopsy with delayed treatment for disease progression) versus immediate treatment (prostatectomy or radiation therapy). Secondary outcomes include preferred management choice (from the four options listed above), diagnosis anxiety, management choice anxiety and management choice at a later time point (for participants who initially choose a monitoring strategy). ETHICS AND DISSEMINATION: Ethics approval has been received from The University of Sydney Human Research Ethics Committee (2023/572). The results of the study will be published in a peer-reviewed medical journal and a plain language summary of the findings will be shared on the Wiser Healthcare publications page http://www.wiserhealthcare.org.au/category/publications/ TRIAL REGISTRATION NUMBERS: Australian New Zealand Clinical Trials Registry (ID 386701 and 386889).
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Neoplasias da Próstata , Humanos , Masculino , Neoplasias da Próstata/diagnóstico , Feminino , Austrália , Antígeno Prostático Específico/sangue , Ansiedade , Ensaios Clínicos Controlados Aleatórios como Assunto , Gradação de Tumores , Pessoa de Meia-Idade , Prostatectomia/métodos , Medição de Risco/métodos , Conduta Expectante/métodosRESUMO
In a previous study, in silico screening of the binding of almost all proteins in the Protein Data Bank to each of the five noble gases xenon, krypton, argon, neon, and helium was reported. This massive and rich data set requires analysis to identify the gas-protein interactions that have the best binding strengths, those where the binding of the noble gas occurs at a site that can modulate the function of the protein, and where this modulation might generate clinically relevant effects. Here, we report a preliminary analysis of this data set using a rational, heuristic score based on binding strength and location. We report a partial prioritized list of xenon protein targets and describe how these data can be analyzed, using arginase and carbonic anhydrase as examples. Our aim is to make the scientific community aware of this massive, rich data set and how it can be analyzed to accelerate future discoveries of xenon-induced biological activity and, ultimately, the development of new "atomic" drugs.
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Proteoma , Xenônio , Criptônio/química , Criptônio/farmacologia , Neônio/farmacologia , Gases Nobres/química , Gases Nobres/metabolismo , Xenônio/química , Xenônio/farmacologiaRESUMO
Patients with Waldenström macroglobulinaemia (WM) are at increased risk of severe COVID-19 infection and have poor immune responses to COVID-19 vaccination. This study assessed whether a closely monitored pause in Bruton's Tyrosine Kinase inhibitor (BTKi) therapy might result in an improved humoral response to a 3rd COVID-19 vaccine dose. Improved response was observed in WM patients who paused their BTKi, compared to a group who did not pause their BTKi. However, the response was attenuated after BTKi recommencement. This data contributes to our understanding of vaccination strategies in this patient group and may help inform consensus approaches in the future.
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Anthozoan chromoproteins are highly pigmented, diversely coloured and readily produced in recombinant expression systems. While they are a versatile and powerful building block in synthetic biology for applications such as biosensor development, they are not widely used in comparison to the related fluorescent proteins, partly due to a lack of structural characterization to aid protein engineering. Here, high-resolution X-ray crystal structures of four open-source chromoproteins, gfasPurple, amilCP, spisPink and eforRed, are presented. These proteins are dimers in solution, and mutation at the conserved dimer interface leads to loss of visible colour development in gfasPurple. The chromophores are trans and noncoplanar in gfasPurple, amilCP and spisPink, while that in eforRed is cis and noncoplanar, and also emits fluorescence. Like other characterized chromoproteins, gfasPurple, amilCP and eforRed contain an sp2-hybridized N-acylimine in the peptide bond preceding the chromophore, while spisPink is unusual and demonstrates a true sp3-hybridized trans-peptide bond at this position. It was found that point mutations at the chromophore-binding site in gfasPurple that substitute similar amino acids to those in amilCP and spisPink generate similar colours. These features and observations have implications for the utility of these chromoproteins in protein engineering and synthetic biology applications.
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Peptídeos , Fluorescência , Proteínas Luminescentes/química , Peptídeos/químicaRESUMO
Lichen associations, a classic model for successful and sustainable interactions between micro-organisms, have been studied for many years. However, there are significant gaps in our understanding about how the lichen symbiosis operates at the molecular level. This review addresses opportunities for expanding current knowledge on signalling and metabolic interplays in the lichen symbiosis using the tools and approaches of systems biology, particularly network modelling. The largely unexplored nature of symbiont recognition and metabolic interdependency in lichens could benefit from applying a holistic approach to understand underlying molecular mechanisms and processes. Together with 'omics' approaches, the application of signalling and metabolic network modelling could provide predictive means to gain insights into lichen signalling and metabolic pathways. First, we review the major signalling and recognition modalities in the lichen symbioses studied to date, and then describe how modelling signalling networks could enhance our understanding of symbiont recognition, particularly leveraging omics techniques. Next, we highlight the current state of knowledge on lichen metabolism. We also discuss metabolic network modelling as a tool to simulate flux distribution in lichen metabolic pathways and to analyse the co-dependence between symbionts. This is especially important given the growing number of lichen genomes now available and improved computational tools for reconstructing such models. We highlight the benefits and possible bottlenecks for implementing different types of network models as applied to the study of lichens.
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A series of novel polyacetylene substituted 2-hydroxy acids and derivatives were prepared and characterized. Alkylation of butane-2,3-diacetal (BDA) protected glycolic acid with iodoalkyl substituted polyacetylene compounds gave the corresponding diacetal protected polyacetylene substituted 2-hydroxy acids. Diacetal deprotection through acid mediated hydrolysis, transesterification or aminolysis afforded the 2-hydroxy-polyacetylenic acid, ester or amide derivatives. Twenty one of these novel compounds were tested against 10 microbes of clinical importance and several of them showed good antimicrobial activity, in particular against Pseudomonas aeruginosa.
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Amidas/química , Antifúngicos/química , Hidroxiácidos/química , Poli-Inos/química , Antifúngicos/síntese química , Antifúngicos/farmacologia , Ésteres , Testes de Sensibilidade Microbiana , Poli-Inos/síntese química , Poli-Inos/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
The synthetic s-triazines are abundant, nitrogen-rich, heteroaromatic compounds used in a multitude of applications including, herbicides, plastics and polymers, and explosives. Their presence in the environment has led to the evolution of bacterial catabolic pathways in bacteria that allow use of these anthropogenic chemicals as a nitrogen source that supports growth. Herbicidal s-triazines have been used since the mid-twentieth century and are among the most heavily used herbicides in the world, despite being withdrawn from use in some areas due to concern about their safety and environmental impact. Bacterial catabolism of the herbicidal s-triazines has been studied extensively. Pseudomonas sp. strain ADP, which was isolated more than thirty years after the introduction of the s-triazine herbicides, has been the model system for most of these studies; however, several alternative catabolic pathways have also been identified. Over the last five years, considerable detail about the molecular mode of action of the s-triazine catabolic enzymes has been uncovered through acquisition of their atomic structures. These structural studies have also revealed insights into the evolutionary origins of this newly acquired metabolic capability. In addition, s-triazine-catabolizing bacteria and enzymes have been used in a range of applications, including bioremediation of herbicides and cyanuric acid, introducing metabolic resistance to plants, and as a novel selectable marker in fermentation organisms. In this review, we cover the discovery and characterization of bacterial strains, metabolic pathways and enzymes that catabolize the s-triazines. We also consider the evolution of these new enzymes and pathways and discuss the practical applications that have been considered for these bacteria and enzymes. One Sentence Summary: A detailed understanding of bacterial herbicide catabolic enzymes and pathways offer new evolutionary insights and novel applied tools.
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Bactérias/enzimologia , Bactérias/genética , Herbicidas/metabolismo , Redes e Vias Metabólicas , Triazinas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biodegradação Ambiental , Evolução Molecular , Regulação Bacteriana da Expressão Gênica , Herbicidas/química , Modelos Biológicos , Conformação Proteica , Pseudomonas/enzimologia , Pseudomonas/genética , Pseudomonas/metabolismo , Triazinas/químicaRESUMO
F420 is a low-potential redox cofactor used by diverse bacteria and archaea. In mycobacteria, this cofactor has multiple roles, including adaptation to redox stress, cell wall biosynthesis, and activation of the clinical antitubercular prodrugs pretomanid and delamanid. A recent biochemical study proposed a revised biosynthesis pathway for F420 in mycobacteria; it was suggested that phosphoenolpyruvate served as a metabolic precursor for this pathway, rather than 2-phospholactate as long proposed, but these findings were subsequently challenged. In this work, we combined metabolomic, genetic, and structural analyses to resolve these discrepancies and determine the basis of F420 biosynthesis in mycobacterial cells. We show that, in whole cells of Mycobacterium smegmatis, phosphoenolpyruvate rather than 2-phospholactate stimulates F420 biosynthesis. Analysis of F420 biosynthesis intermediates present in M. smegmatis cells harboring genetic deletions at each step of the biosynthetic pathway confirmed that phosphoenolpyruvate is then used to produce the novel precursor compound dehydro-F420-0. To determine the structural basis of dehydro-F420-0 production, we solved high-resolution crystal structures of the enzyme responsible (FbiA) in apo-, substrate-, and product-bound forms. These data show the essential role of a single divalent cation in coordinating the catalytic precomplex of this enzyme and demonstrate that dehydro-F420-0 synthesis occurs through a direct substrate transfer mechanism. Together, these findings resolve the biosynthetic pathway of F420 in mycobacteria and have significant implications for understanding the emergence of antitubercular prodrug resistance.IMPORTANCE Mycobacteria are major environmental microorganisms and cause many significant diseases, including tuberculosis. Mycobacteria make an unusual vitamin-like compound, F420, and use it to both persist during stress and resist antibiotic treatment. Understanding how mycobacteria make F420 is important, as this process can be targeted to create new drugs to combat infections like tuberculosis. In this study, we show that mycobacteria make F420 in a way that is different from other bacteria. We studied the molecular machinery that mycobacteria use to make F420, determining the chemical mechanism for this process and identifying a novel chemical intermediate. These findings also have clinical relevance, given that two new prodrugs for tuberculosis treatment are activated by F420.
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Chemosensory protein based olfactory biosensors are expected to play a significant role in next-generation volatile organic compound (VOC) detection systems due to their ultra-high sensitivity and selectivity. As these biosensors can perform most efficiently in aqueous environments, the detection systems need to incorporate a gas sampling interface for gas-to-liquid extraction. This interface should extract the VOCs from the gas phase with high efficiency and transfer them into the liquid containing biosensors to enable subsequent detection. To design such a transfer interface, an understanding of the key parameters influencing the gas-to-liquid extraction efficiency of target VOCs is crucial. This paper reports a gas sampling interface system based on a microfluidic open-channel device for gas-to-liquid extraction. By using this device as a model platform, the key parameters dictating the VOC extraction efficiency were identified. When loaded with 30 µL of capture liquid, the microfluidic device generates a gas-liquid interface area of 3 cm2 without using an interfacial membrane. The pumpless operation based on capillary flow was demonstrated for capture liquid loading and collection. Gas samples spiked with lipophilic model volatiles (hexanal and allyl methyl sulfide) were used for characterization of the VOC extraction efficiency. Decreasing the sampling temperature to 15 °C had a significant impact on increasing capture efficiency, while variation in the gas sampling flow rate had no significant impact in the range between 40-120 mL min-1. This study found more than a 10-fold increase in capture efficiency by chemical modification of the capture liquid with alpha-cyclodextrin. The highest capture efficiency of 30% was demonstrated with gas samples spiked with hexanal to a concentration of 16 ppm (molar proportion). The approach in this study should be useful for further optimisation of miniaturised gas-to-liquid extraction systems and contribute to the design of chemosensory protein-based VOC detection systems.
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Protein engineering strategies are often guided by our understanding of how the structure of a protein determines its function. However, our understanding is generally restricted to small regions of a protein, namely the active site and its immediate vicinity, while the remainder of the protein is something of an enigma. Studying highly homologous transaminases with strictly conserved active sites, but different substrate preferences and activities, we predict and experimentally validate that the surface of the protein far from the active site carries out a decisive role in substrate selectivity and catalytic efficiency. Using a unique molecular dynamics approach and novel trajectory analysis, we demonstrate the phenomenon of surface-directed ligand diffusion in this well-known protein family for the first time. Further, we identify the residues involved in directing substrate, design surface channel variants endowed for improved kinetic properties and establish a broadly applicable new approach for protein engineering.