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1.
Microbiol Res ; 268: 127279, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36592576

RESUMO

Solidesulfovibrio fructosivorans (formely Desulfovibrio fructosovorans), an anaerobic sulfate-reducing bacterium, possesses six gene clusters encoding six hydrogenases catalyzing the reversible oxidation of hydrogen gas (H2) into protons and electrons. One of these, named Hnd, was demonstrated to be an electron-bifurcating hydrogenase Hnd (Kpebe et al., 2018). It couples the exergonic reduction of NAD+ to the endergonic reduction of a ferredoxin with electrons derived from H2 and whose function has been recently shown to be involved in ethanol production under pyruvate fermentation (Payne 2022). To understand further the physiological role of Hnd in S. fructosivorans, we compared the mutant deleted of part of the hnd gene with the wild-type strain grown on pyruvate without sulfate using NMR-based metabolomics. Our results confirm that Hnd is profoundly involved in ethanol metabolism, but also indirectly intervenes in global carbon metabolism and additional metabolic processes such as the biosynthesis of branched-chain amino acids. We also highlight the metabolic reprogramming induced by the deletion of hndD that leads to the upregulation of several NADP-dependent pathways.


Assuntos
Hidrogenase , Hidrogenase/genética , Hidrogenase/química , Hidrogenase/metabolismo , Fermentação , Elétrons , Ácido Pirúvico , Oxirredução , Hidrogênio/metabolismo
2.
Sci Rep ; 12(1): 22232, 2022 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-36564432

RESUMO

The mytilid mussel Bathymodiolus thermophilus lives in the deep-sea hydrothermal vent regions due to its relationship with chemosynthetic symbiotic bacteria. It is well established that symbionts reside in the gill bacteriocytes of the mussel and can utilize hydrogen sulfide, methane, and hydrogen from the surrounding environment. However, it is observed that some mussel symbionts either possess or lack genes for hydrogen metabolism within the single-ribotype population and host mussel species level. Here, we found a hydrogenase cluster consisting of additional H2-sensing hydrogenase subunits in a complete genome of B. thermophilus symbiont sampled from an individual mussel from the East Pacific Rise (EPR9N). Also, we found methylated regions sparsely distributed throughout the EPR9N genome, mainly in the transposase regions and densely present in the rRNA gene regions. CRISPR diversity analysis confirmed that this genome originated from a single symbiont strain. Furthermore, from the comparative analysis, we observed variation in genome size, gene content, and genome re-arrangements across individual hosts suggesting multiple symbiont strains can associate with B. thermophilus. The ability to acquire locally adaptive various symbiotic strains may serve as an effective mechanism for successfully colonizing different chemosynthetic environments across the global oceans by host mussels.


Assuntos
Hidrogenase , Fontes Hidrotermais , Mytilidae , Animais , Hidrogenase/genética , Hidrogenase/metabolismo , Mytilidae/genética , Bactérias , Metano/metabolismo , Família Multigênica , Simbiose/genética , Brânquias/microbiologia
3.
Chem Commun (Camb) ; 58(87): 12168-12171, 2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-36239209

RESUMO

Despite a variety of [Fe]-H2ase models prepared so far, the structural and functional modeling study of the enzyme has remained a great challenge. Now, we report a new type of flexible pyridine ligand (FPL)-based synthetic method by which two novel [Fe]-H2ase models have been prepared. Notably, the two models contain not only a biomimetic fac-acyl C, pyridyl N, thioether S coordination mode but also possess the enzyme-like H2/D2 activation functions.


Assuntos
Materiais Biomiméticos , Hidrogenase , Proteínas Ferro-Enxofre , Hidrogenase/química , Ligantes , Biomimética , Modelos Moleculares , Proteínas Ferro-Enxofre/química , Piridinas/química , Materiais Biomiméticos/química
4.
J Phys Chem B ; 126(43): 8777-8790, 2022 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-36269122

RESUMO

Hydrogenases are a group of enzymes that have caught the interest of researchers in renewable energies, due to their ability to catalyze the redox reaction of hydrogen. The exploitation of hydrogenases in electrochemical devices requires their immobilization on the surface of suitable electrodes, such as graphite. The orientation of the enzyme on the electrode is important to ensure a good flux of electrons to the catalytic center, through an array of iron-sulfur clusters. Here we present a computational approach to determine the possible orientations of a [NiFe] hydrogenase (PDB 1e3d) on a planar electrode, as a function of pH, salinity, and electrode potential. The calculations are based on the solution of the linearized Poisson-Boltzmann equation, using the PyGBe software. The results reveal that electrostatic interactions do not truly immobilize the enzyme on the surface of the electrode, but there is instead a dynamic equilibrium between different orientations. Nonetheless, after averaging over all thermally accessible orientations, we find significant differences related to the solution's salinity and pH, while the effect of the electrode potential is relatively weak. We also combine models for the protein adsoption-desorption equilibria and for the electron transfer between the proteins and the electrode to arrive at a prediction of the electrode's activity as a function of the enzyme concentration.


Assuntos
Hidrogenase , Hidrogenase/metabolismo , Eletricidade Estática , Eletrodos , Hidrogênio/metabolismo , Transporte de Elétrons , Oxirredução , Proteínas/metabolismo
5.
Angew Chem Int Ed Engl ; 61(50): e202213239, 2022 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-36264001

RESUMO

In the biosynthesis of the iron-guanylylpyridinol (FeGP) cofactor, 6-carboxymethyl-5-methyl-4-hydroxy-2-pyridinol (1) is 3-methylated to form 2, then 4-guanylylated to form 3, and converted into the full cofactor. HcgA-G proteins catalyze the biosynthetic reactions. Herein, we report the function of two radical S-adenosyl methionine enzymes, HcgA and HcgG, as uncovered by in vitro complementation experiments and the use of purified enzymes. In vitro biosynthesis using the cell extract from the Methanococcus maripaludis ΔhcgA strain was complemented with HcgA or precursors 1, 2 or 3. The results suggested that HcgA catalyzes the biosynthetic reaction that forms 1. We demonstrated the formation of 1 by HcgA using the 3 kDa cell extract filtrate as the substrate. Biosynthesis in the ΔhcgG system was recovered by HcgG but not by 3, which indicated that HcgG catalyzes the reactions after the biosynthesis of 3. The data indicated that HcgG contributes to the formation of CO and completes biosynthesis of the FeGP cofactor.


Assuntos
Hidrogenase , Proteínas Ferro-Enxofre , Hidrogenase/metabolismo , Extratos Celulares , Proteínas Ferro-Enxofre/metabolismo , S-Adenosilmetionina/metabolismo , Ferro/metabolismo
6.
Biochemistry (Mosc) ; 87(10): 1098-1108, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36273878

RESUMO

Light-dependent hydrogen production by microalgae attracts attention of researchers because of the potential practical application. It is generally recognized that Calvin-Benson-Bassham cycle competes with hydrogen production process for electrons, and substrate (CO2) limitation of the cycle can increase hydrogen production rate. Furthermore, photosystem II is not destroyed by CO2 deficiency. We studied photoautotrophic cultures of Chlamydomonas reimhardtii under CO2 deficiency. Under the flow of air with removed CO2 the cultures reached stationary phase of growth and the photosystem II was downregulated due to overreduction of plastoquinone pool followed by degradation of the entire photosynthetic machinery. Under the Ar flow in the absence of CO2 the cultures were brought to microaerobic conditions producing small amounts of hydrogen (5 ml H2 day-1 liter-1 culture). Similar to the case of incubation under air atmosphere, prolonged incubation of cultures under microaerobic conditions resulted in down-regulation of photosystem II due to overreduction of plastoquinone pool with following degradation of whole photosynthetic machinery. Following removal of CO2, transfer of cultures into dark anaerobic conditions (2.5 h), and illumination with low-intensity light resulted in the cultures producing H2 with high initial rate. Total microalgal hydrogen production under these conditions was 56 ml H2 liter-1 culture. Thus, the CO2-deprived photoautotrophic cultures produce hydrogen. Hydrogen production was limited by the toxic effect of oxygen on hydrogenase but not by the Calvin-Benson-Bassham cycle competition with hydrogen production process.


Assuntos
Chlamydomonas reinhardtii , Hidrogenase , Chlamydomonas reinhardtii/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Dióxido de Carbono/metabolismo , Hidrogenase/metabolismo , Hidrogenase/farmacologia , Plastoquinona/farmacologia , Enxofre/metabolismo , Enxofre/farmacologia , Fotossíntese/fisiologia , Hidrogênio , Oxigênio/metabolismo
7.
Curr Microbiol ; 79(12): 360, 2022 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-36253650

RESUMO

The Hyf-type formate hydrogen lyase (FHL) complex was first proposed based on sequence comparisons in Escherichia coli in 1997 (Andrews et al. in Microbiology 143:3633-3647, 1997). The hydrogenase in the Hyf-type FHL was estimated to be a proton-translocating energy-conserving [NiFe]-hydrogenase. Although the structure of FHL is similar to that of complex I, silent gene expression in E. coli has caused delays in unveiling the genetic and biochemical features of the FHL. The entire set of genes required for Hyf-type FHL synthesis has also been found in the genome sequences of Vibrio tritonius in 2015 (Matsumura et al. in Int J Hydrog Energy 40:9137-9146, 2015), which produces more hydrogen (H2) than E. coli. Here we investigate the physiological characteristics, genome comparisons, and gene expressions to elucidate the genetic backgrounds of Hyf-type FHL, and how Hyf-type FHL correlates with the higher H2 production of V. tritonius. Physiological comparisons among the seven H2-producing vibrios reveal that V. porteresiae and V. tritonius, grouped in the Porteresiae clade, show greater capacity for H2 production than the other species. The structures of FHL-Hyp gene clusters were closely related in both Porteresiae species, but differed from those of the other species with the presence of hupE, a possible nickel permease gene. Interestingly, deeper genome comparisons revealed the co-presence of nickel ABC transporter genes (nik) with the Hyf-type FHL gene only on the genome of the Porteresiae clade species. Therefore, active primary Ni transport might be one of the key factors characterizing higher H2 production in V. tritonius. Furthermore, the expression of FHL gene cluster was significantly up-regulated in V. tritonius cells stimulated with formate, indicating that formate is likely to be a control factor for the gene expression of V. tritonius FHL in a similar way to the formate regulon encoding the E. coli FHL.


Assuntos
Hidrogenase , Vibrio , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Escherichia coli/fisiologia , Proteínas de Escherichia coli/genética , Formiatos/metabolismo , Genômica , Hidrogênio/metabolismo , Hidrogenase/genética , Hidrogenase/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Níquel/metabolismo , Vibrio/genética , Vibrio/fisiologia
8.
Int J Mol Sci ; 23(19)2022 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-36233092

RESUMO

Soil alkalinity is an important stressor that impairs crop growth and development, resulting in reduced crop productivity. Unlike salinity stress, research efforts to understand the mechanism of plant adaptation to alkaline stress is limited in rice, a major staple food for the world population. We evaluated a population of 193 recombinant inbred lines (RIL) developed from a cross between Cocodrie and N22 under alkaline stress at the seedling stage. Using a linkage map consisting of 4849 SNP markers, 42 additive QTLs were identified. There were seven genomic regions where two or more QTLs for multiple traits colocalized. Three important QTL clusters were targeted, and several candidate genes were identified based on high impact variants using whole genome sequences (WGS) of both parents and differential expression in response to alkalinity stress. These genes included two expressed protein genes, the glucan endo-1,3-beta-glucosidase precursor, F-box domain-containing proteins, double-stranded RNA-binding motif-containing protein, aquaporin protein, receptor kinase-like protein, semialdehyde hydrogenase, and NAD-binding domain-containing protein genes. Tolerance to alkaline stress in Cocodrie was most likely due to the low Na+/K+ ratio resulting from reduced accumulation of Na+ ions and higher accumulation of K+ in roots and shoots. Our study demonstrated the utility of integrating QTL mapping with WGS to identify the candidate genes in the QTL regions. The QTLs and candidate genes originating from the tolerant parent Cocodrie should be targeted for introgression to improve alkalinity tolerance in rice and to elucidate the molecular basis of alkali tolerance.


Assuntos
Celulases , Hidrogenase , Oryza , Álcalis , Celulases/genética , Glucanos , Hidrogenase/genética , NAD/genética , RNA de Cadeia Dupla , Solo , Sequenciamento Completo do Genoma
9.
Phys Chem Chem Phys ; 24(40): 24767-24783, 2022 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-36200672

RESUMO

Ultrafast two-dimensional infrared (2D-IR) spectroscopy of Escherichia coli Hyd-1 (EcHyd-1) reveals the structural and dynamic influence of the protein scaffold on the Fe(CO)(CN)2 unit of the active site. Measurements on as-isolated EcHyd-1 probed a mixture of active site states including two, which we assign to Nir-SI/II, that have not been previously observed in the E. coli enzyme. Explicit assignment of carbonyl (CO) and cyanide (CN) stretching bands to each state is enabled by 2D-IR. Energies of vibrational levels up to and including two-quantum vibrationally excited states of the CO and CN modes have been determined along with the associated vibrational relaxation dynamics. The carbonyl stretching mode potential is well described by a Morse function and couples weakly to the cyanide stretching vibrations. In contrast, the two CN stretching modes exhibit extremely strong coupling, leading to the observation of formally forbidden vibrational transitions in the 2D-IR spectra. We show that the vibrational relaxation times and structural dynamics of the CO and CN ligand stretching modes of the enzyme active site differ markedly from those of a model compound K[CpFe(CO)(CN)2] in aqueous solution and conclude that the protein scaffold creates a unique biomolecular environment for the NiFe site that cannot be represented by analogy to simple models of solvation.


Assuntos
Hidrogenase , Hidrogenase/química , Domínio Catalítico , Escherichia coli/metabolismo , Ligantes , Cianetos/química , Espectrofotometria Infravermelho/métodos , Proteínas
10.
Microbiology (Reading) ; 168(10)2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36197793

RESUMO

During enterobacterial mixed-acid fermentation, formate is generated from pyruvate by the glycyl-radical enzyme pyruvate formate-lyase (PflB). In Escherichia coli, especially at low pH, formate is then disproportionated to CO2 and H2 by the cytoplasmically oriented, membrane-associated formate hydrogenlyase (FHL) complex. If electron acceptors are available, however, formate is oxidized by periplasmically oriented, respiratory formate dehydrogenases. Formate translocation across the cytoplasmic membrane is controlled by the formate channel, FocA, a member of the formate-nitrite transporter (FNT) family of homopentameric anion channels. This review highlights recent advances in our understanding of how FocA helps to maintain intracellular formate and pH homeostasis during fermentation. Efflux and influx of formate/formic acid are distinct processes performed by FocA and both are controlled through protein interaction between FocA's N-terminal domain with PflB. Formic acid efflux by FocA helps to maintain cytoplasmic pH balance during exponential-phase growth. Uptake of formate against the electrochemical gradient (inside negative) is energetically and mechanistically challenging for a fermenting bacterium unless coupled with proton/cation symport. Translocation of formate/formic acid into the cytoplasm necessitates an active FHL complex, whose synthesis also depends on formate. Thus, FocA, FHL and PflB function together to govern formate homeostasis. We explain how FocA achieves efflux of formic acid and propose mechanisms for pH-dependent uptake of formate both with and without proton symport. We propose that FocA displays both channel- and transporter-like behaviour. Whether this translocation behaviour is shared by other members of the FNT family is also discussed.


Assuntos
Proteínas de Escherichia coli , Hidrogenase , Ânions/metabolismo , Dióxido de Carbono/metabolismo , Enterobacteriaceae/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Formiato Desidrogenases/genética , Formiato Desidrogenases/metabolismo , Formiatos/metabolismo , Homeostase , Concentração de Íons de Hidrogênio , Hidrogenase/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Nitritos/metabolismo , Prótons , Piruvatos/metabolismo
11.
J Am Chem Soc ; 144(44): 20267-20277, 2022 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-36305871

RESUMO

A new method to install a proton relay that enhances the reactivity near an active catalytic site for H2 production is reported, afforded by the electrochemical reduction and protonation of one of the ligands in the paddlewheel Rh2(II,II) hydrogen evolution complex, cis-[Rh2(DPhF)2(bncn)2]2+ (Rh-bncn; DPhF = N,N'-diphenylformamidinate, bncn = benzo[c]cinnoline). An electrochemical reversible prewave is observed for Rh-bncn at potentials more positive than the first bncn-centered reduction couple in the presence of strong acids, observed at -0.72 V vs Fc+/0 (Fc = ferrocene) in the cyclic voltammograms (CVs) in DMF (0.1 M TBAPF6). The origin of this prewave is shown to arise from a precatalytic transformation that originates from a concerted proton-electron transfer (CPET) event occurring at one of the bridging bncn ligands. Through electrochemical analysis, CV simulations, and electronic structure calculations, a reaction mechanism is elucidated. In this system, the electrochemically formed N-H bond on the reduced bncn ligand serves as a proton relay in the H2 formation reaction through a cooperative interligand pathway involving one of the bridging DPhF ligands after a second reduction step, accessible at approximately -1.15 V vs Fc+/0. Since calculations show that hydrogen evolution takes place at the bridging ligands and does not involve the dirhodium core, it is predicted that more abundant metal centers can be incorporated into this ligand scaffold, leading to new candidates for electrocatalytic hydrogen reduction. As such, this work delineates a new design strategy to incorporate proton relays in molecular bimetallic hydrogen evolution electrocatalysts to achieve higher efficiency.


Assuntos
Hidrogênio , Hidrogenase , Hidrogênio/química , Prótons , Hidrogenase/química , Ligantes , Catálise
12.
Metallomics ; 14(10)2022 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-36190308

RESUMO

[NiFe]-hydrogenases are used by several human pathogens to catalyze the reversible conversion between molecular hydrogen and protons and electrons. Hydrogenases provide an increased metabolic flexibility for pathogens, such as Escherichia coli and Helicobacter pylori, by allowing the use of molecular hydrogen as an energy source to promote survival in anaerobic environments. With the rise of antimicrobial resistance and the desire for novel therapeutics, the [NiFe]-hydrogenases are alluring targets. Inhibiting the nickel insertion pathway of [NiFe]-hydrogenases is attractive as this pathway is required for the generation of functional enzymes and is orthogonal to human biochemistry. In this work, nickel availability for the production and function of E. coli [NiFe]-hydrogenase was explored through immunoblot and activity assays. Whole-cell hydrogenase activities were assayed in high throughput against a small molecule library of known bioactives. Iodoquinol was identified as a potential inhibitor of the nickel biosynthetic pathway of [NiFe]-hydrogenase through a two-step screening process, but further studies with immunoblot assays showed confounding effects dependent on the cell growth phase. This study highlights the significance of considering the growth phenotype for whole-cell based assays overall and its effects on various cellular processes influenced by metal trafficking and homeostasis.


Assuntos
Anti-Infecciosos , Hidrogenase , Escherichia coli/metabolismo , Humanos , Hidrogênio/metabolismo , Hidrogenase/metabolismo , Iodoquinol , Níquel/metabolismo , Prótons
13.
Mol Biol Evol ; 39(10)2022 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-36181435

RESUMO

Thermoprofundales, formerly Marine Benthic Group D (MBG-D), is a ubiquitous archaeal lineage found in sedimentary environments worldwide. However, its taxonomic classification, metabolic pathways, and evolutionary history are largely unexplored because of its uncultivability and limited number of sequenced genomes. In this study, phylogenomic analysis and average amino acid identity values of a collection of 146 Thermoprofundales genomes revealed five Thermoprofundales subgroups (A-E) with distinct habitat preferences. Most of the microorganisms from Subgroups B and D were thermophiles inhabiting hydrothermal vents and hot spring sediments, whereas those from Subgroup E were adapted to surface environments where sunlight is available. H2 production may be featured in Thermoprofundales as evidenced by a gene cluster encoding the ancient membrane-bound hydrogenase (MBH) complex. Interestingly, a unique structure separating the MBH gene cluster into two modular units was observed exclusively in the genomes of Subgroup E, which included a peripheral arm encoding the [NiFe] hydrogenase domain and a membrane arm encoding the Na+/H+ antiporter domain. These two modular structures were confirmed to function independently by detecting the H2-evolving activity in vitro and salt tolerance to 0.2 M NaCl in vivo, respectively. The peripheral arm of Subgroup E resembles the proposed common ancestral respiratory complex of modern respiratory systems, which plays a key role in the early evolution of life. In addition, molecular dating analysis revealed that Thermoprofundales is an early emerging archaeal lineage among the extant MBH-containing microorganisms, indicating new insights into the evolution of this ubiquitous archaea lineage.


Assuntos
Archaea , Hidrogenase , Archaea/genética , Archaea/metabolismo , Hidrogenase/química , Hidrogenase/genética , Hidrogenase/metabolismo , Cloreto de Sódio/metabolismo , Filogenia , Sistema Respiratório/metabolismo , Aminoácidos/genética , Antiporters/genética , Antiporters/metabolismo
14.
Angew Chem Int Ed Engl ; 61(49): e202212074, 2022 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-36137942

RESUMO

The [FeFe]-hydrogenase H-cluster is a complex organometallic cofactor whose assembly and installation requires three dedicated accessory proteins referred to as HydE, HydF, and HydG. The roles of these maturases and the precise mechanisms by which they synthesize and insert the H-cluster are not fully understood. This Minireview will focus on new insights into the [FeFe]-hydrogenase maturation process that have been provided by in vitro approaches in which the biosynthetic pathway has been partially or fully reconstructed using semisynthetic and enzyme-based approaches. Specifically, the application of these in vitro, semisynthetic, and fully defined approaches has shed light on the roles of individual maturation enzymes, the nature of H-cluster assembly intermediates, the molecular precursors of H-cluster ligands, and the sequence of steps involved in [FeFe]-hydrogenase maturation.


Assuntos
Hidrogenase , Proteínas Ferro-Enxofre , Hidrogenase/metabolismo , Proteínas Ferro-Enxofre/metabolismo
15.
Arch Microbiol ; 204(10): 627, 2022 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-36114886

RESUMO

Although Escherichia coli has four hydrogenases, their definite roles in fermentation are still not clear. In this study, all the operon deletion mutants of E.coli hydrogenases (∆hya, ∆hyb, ∆hyc, or ∆hyf) were constructed to evaluate the hydrogen metabolism in comparison to their respective single-gene deletion mutants of large subunits (∆hyaB, ∆hybC, ∆hycE, and ∆hyfG). Besides the hyc operon mutant that expectedly showed no hydrogen synthesis, the hyb operon mutant showed low hydrogen production and demonstrated significantly reduced growth under anaerobic conditions. The present work also provided first-hand data where deleterious effects of operon deletion were compared with single-gene deletion mutations and the results showed that the former type of deletion was found to cause more prominent phenotypic effects than the latter one. Interestingly, hyb operon mutant was remarkably distinct from other operon mutants, specifically in its inability to utilize glucose under both aerobic and anaerobic conditions. Further studies on this mutant revealed a significant reduction of the total intracellular ATP and NADH concentrations, which could explain its impaired glucose metabolism. In this way, Hyd-2 was verified as crucial not only in glucose metabolism but also in energy balance and redox homeostasis of the cells. Furthermore, a decreased expression of glucose metabolism-associated genes, particularly ppc and pykA, indicated their regulation by hyb operon, and thereby, glucose consumption. Moreover, the transcriptional changes in this mutant indicated the wide genomic connectivity of hyb operon to other metabolisms.


Assuntos
Escherichia coli/enzimologia , Hidrogenase , Trifosfato de Adenosina/metabolismo , Escherichia coli/genética , Glucose/metabolismo , Hidrogenase/genética , Hidrogenase/metabolismo , NAD/metabolismo , Óperon
16.
Chemosphere ; 309(Pt 1): 136535, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36150484

RESUMO

The biogas production (BP), volatile fatty acids (VFAs), microbial communities, and microbes' active enzymes were studied upon the addition of biochar (0-1.5%) at 6% and 8% slaughterhouse waste (SHW) loadings. The 0.5% biochar enhanced BP by 1.5- and 1.6-folds in 6% and 8% SHW-loaded reactors, respectively. Increasing the biochar up to 1.5% caused a reduction in BP at 6% SHW. However, the BP from 8% of SHW was enhanced by 1.4-folds at 1.5% biochar. The VFAs production in all 0.5% biochar amended reactors was highly significant compared to control (p-value < 0.05). The biochar addition increased the bacterial and archaeal diversity at both 6% and 8% SHW loadings. The highest number of OTUs at 0.5% biochar were 567 and 525 in 6% and 8% SHW, respectively. Biochar prompted the Clostridium abundance and increased the lyases and transaminases involved in the degradation of lipids and protein, respectively. Biochar addition improved the Methanosaeta and Methanosphaera abundance in which the major enzymes were reductase and hydrogenase. The archaeal enzymes showed mixed acetoclastic and hydrogenotrophic methanogenesis.


Assuntos
Hidrogenase , Liases , Microbiota , Archaea/metabolismo , Biocombustíveis , Anaerobiose , Reatores Biológicos , Matadouros , Metano/metabolismo , Hidrogenase/metabolismo , Bactérias/metabolismo , Ácidos Graxos Voláteis/metabolismo , Liases/metabolismo , Transaminases , Digestão
17.
Medicina (Kaunas) ; 58(9)2022 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-36143845

RESUMO

Background and Objective: Helicobacter pylori is a human-stomach-dwelling organism that causes many gastric illnesses, including gastritis, ulcer, and gastric cancer. The purpose of the study was to perform differential proteomic analysis on H. pylori isolates from gastritis, ulcer, and gastric cancer patients. Materials and Methods: H. pylori was isolated from antrum and fundus biopsies obtained from patients who visited the Department of Gastroenterology. Using nano-LC-QTOF MS/MS analysis, differentially regulated proteins were identified through proteome profiling of pooled samples of H. pylori isolated from gastritis, ulcer, and gastric cancer patients. Antigenic scores and cellular localization of proteins were determined using additional prediction tools. Results: A total of 14 significantly regulated proteins were identified in H. pylori isolated from patients with either gastritis, ulcer, or gastric cancer. Comparative analysis of groups revealed that in the case of cancer vs. gastritis, six proteins were overexpressed, out of which two proteins, including hydrogenase maturation factor (hypA) and nucleoside diphosphate kinase (ndk) involved in bacterial colonization, were only upregulated in isolates from cancer patients. Similarly, in cancer vs. ulcer, a total of nine proteins were expressed. Sec-independent protein translocase protein (tatB), involved in protein translocation, and pseudaminic acid synthase I (pseI), involved in the synthesis of functional flagella, were upregulated in cancer, while hypA and ndk were downregulated. In ulcer vs. gastritis, eight proteins were expressed. In this group, tatB was overexpressed. A reduction in thioredoxin peroxidase (bacterioferritin co-migratory protein (bcp)) was observed in ulcer vs. gastritis and cancer vs. ulcer. Conclusion: Our study suggested three discrete protein signatures, hypA, tatB, and bcp, with differential expression in gastritis, ulcer, and cancer. Protein expression profiles of H. pylori isolated from patients with these gastric diseases will help to understand the virulence and pathogenesis of H. pylori.


Assuntos
Gastrite , Infecções por Helicobacter , Helicobacter pylori , Hidrogenase , Núcleosídeo-Difosfato Quinase , Neoplasias Gástricas , Gastrite/microbiologia , Glicogênio Sintase/metabolismo , Infecções por Helicobacter/microbiologia , Humanos , Hidrogenase/metabolismo , Núcleosídeo-Difosfato Quinase/metabolismo , Paquistão , Peroxirredoxinas/metabolismo , Proteoma/metabolismo , Proteômica , Neoplasias Gástricas/patologia , Espectrometria de Massas em Tandem , Úlcera
18.
Nat Commun ; 13(1): 5395, 2022 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-36104349

RESUMO

The prototypical hydrogen-producing enzyme, the membrane-bound formate hydrogenlyase (FHL) complex from Escherichia coli, links formate oxidation at a molybdopterin-containing formate dehydrogenase to proton reduction at a [NiFe] hydrogenase. It is of intense interest due to its ability to efficiently produce H2 during fermentation, its reversibility, allowing H2-dependent CO2 reduction, and its evolutionary link to respiratory complex I. FHL has been studied for over a century, but its atomic structure remains unknown. Here we report cryo-EM structures of FHL in its aerobically and anaerobically isolated forms at resolutions reaching 2.6 Å. This includes well-resolved density for conserved loops linking the soluble and membrane arms believed to be essential in coupling enzymatic turnover to ion translocation across the membrane in the complex I superfamily. We evaluate possible structural determinants of the bias toward hydrogen production over its oxidation and describe an unpredicted metal-binding site near the interface of FdhF and HycF subunits that may play a role in redox-dependent regulation of FdhF interaction with the complex.


Assuntos
Proteínas de Escherichia coli , Hidrogenase , Escherichia coli , Proteínas de Escherichia coli/química , Formiato Desidrogenases/química , Hidrogênio , Hidrogenase/química
19.
Microb Cell Fact ; 21(1): 193, 2022 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-36123684

RESUMO

BACKGROUND: O2-tolerant [NiFe]-hydrogenases offer tremendous potential for applications in H2-based technology. As these metalloenzymes undergo a complicated maturation process that requires a dedicated set of multiple accessory proteins, their heterologous production is challenging, thus hindering their fundamental understanding and the development of related applications. Taking these challenges into account, we selected the comparably simple regulatory [NiFe]-hydrogenase (RH) from Cupriavidus necator as a model for the development of bioprocesses for heterologous [NiFe]-hydrogenase production. We already reported recently on the high-yield production of catalytically active RH in Escherichia coli by optimizing the culture conditions in shake flasks. RESULTS: In this study, we further increase the RH yield and ensure consistent product quality by a rationally designed high cell density fed-batch cultivation process. Overall, the bioreactor cultivations resulted in ˃130 mg L-1 of catalytically active RH which is a more than 100-fold increase compared to other RH laboratory bioreactor scale processes with C. necator. Furthermore, the process shows high reproducibility of the previously selected optimized conditions and high productivity. CONCLUSIONS: This work provides a good opportunity to readily supply such difficult-to-express complex metalloproteins economically and at high concentrations to meet the demand in basic and applied studies.


Assuntos
Hidrogenase , Metaloproteínas , Reatores Biológicos , Contagem de Células , Escherichia coli , Hidrogenase/metabolismo , Metaloproteínas/metabolismo , Reprodutibilidade dos Testes
20.
J Am Chem Soc ; 144(37): 17022-17032, 2022 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-36084022

RESUMO

NAD+-reducing [NiFe] hydrogenases are valuable biocatalysts for H2-based energy conversion and the regeneration of nucleotide cofactors. While most hydrogenases are sensitive toward O2 and elevated temperatures, the soluble NAD+-reducing [NiFe] hydrogenase from Hydrogenophilus thermoluteolus (HtSH) is O2-tolerant and thermostable. Thus, it represents a promising candidate for biotechnological applications. Here, we have investigated the catalytic activity and active-site structure of native HtSH and variants in which a glutamate residue in the active-site cavity was replaced by glutamine, alanine, and aspartate. Our biochemical, spectroscopic, and theoretical studies reveal that at least two active-site states of oxidized HtSH feature an unusual architecture in which the glutamate acts as a terminal ligand of the active-site nickel. This observation demonstrates that crystallographically observed glutamate coordination represents a native feature of the enzyme. One of these states is diamagnetic and characterized by a very high stretching frequency of an iron-bound active-site CO ligand. Supported by density-functional-theory calculations, we identify this state as a high-valent species with a biologically unprecedented formal Ni(IV) ground state. Detailed insights into its structure and dynamics were obtained by ultrafast and two-dimensional infrared spectroscopy, demonstrating that it represents a conformationally strained state with unusual bond properties. Our data further show that this state is selectively and reversibly formed under oxic conditions, especially upon rapid exposure to high O2 levels. We conclude that the kinetically controlled formation of this six-coordinate high-valent state represents a specific and precisely orchestrated stereoelectronic response toward O2 that could protect the enzyme from oxidative damage.


Assuntos
Hidrogenase , Alanina/metabolismo , Ácido Aspártico/metabolismo , Domínio Catalítico , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Hidrogenase/química , Hydrogenophilaceae , Ferro/química , Ligantes , NAD/metabolismo , Níquel/química , Oxirredução , Oxigênio/química
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