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1.
Molecules ; 26(12)2021 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-34204747

RESUMO

Cytochrome P450s (P450) are important enzymes in biology with useful biochemical reactions in, for instance, drug and xenobiotics metabolisms, biotechnology, and health. Recently, the crystal structure of a new member of the CYP116B family has been resolved. This enzyme is a cytochrome P450 (CYP116B46) from Tepidiphilus thermophilus (P450-TT) and has potential for the oxy-functionalization of organic molecules such as fatty acids, terpenes, steroids, and statins. However, it was thought that the opening to its hitherto identified substrate channel was too small to allow organic molecules to enter. To investigate this, we performed molecular dynamics simulations on the enzyme. The results suggest that the crystal structure is not relaxed, possibly due to crystal packing effects, and that its tunnel structure is constrained. In addition, the simulations revealed two key amino acid residues at the mouth of the channel; a glutamyl and an arginyl. The glutamyl's side chain tightens and relaxes the opening to the channel in conjunction with the arginyl's, though the latter's side chain is less dramatically changed after the initial relaxation of its conformations. Additionally, it was observed that the effect of increased temperature did not considerably affect the dynamics of the enzyme fold, including the relative solvent accessibility of the amino acid residues that make up the substrate channel wall even as compared to the changes that occurred at room temperature. Interestingly, the substrate channel became distinguishable as a prominent tunnel that is likely to accommodate small- to medium-sized organic molecules for bioconversions. That is, P450-TT has the ability to pass appropriate organic substrates to its active site through its elaborate substrate channel, and notably, is able to control or gate any molecules at the opening to this channel.


Assuntos
Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Hydrogenophilaceae/metabolismo , Sítios de Ligação/fisiologia , Domínio Catalítico/fisiologia , Cristalografia por Raios X/métodos , Sistema Enzimático do Citocromo P-450/fisiologia , Hydrogenophilaceae/enzimologia , Simulação de Dinâmica Molecular , Oxirredução , Ligação Proteica/fisiologia , Especificidade por Substrato/fisiologia
2.
Nat Commun ; 12(1): 4028, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34188059

RESUMO

CNNM/CorB proteins are a broadly conserved family of integral membrane proteins with close to 90,000 protein sequences known. They are associated with Mg2+ transport but it is not known if they mediate transport themselves or regulate other transporters. Here, we determine the crystal structure of an archaeal CorB protein in two conformations (apo and Mg2+-ATP bound). The transmembrane DUF21 domain exists in an inward-facing conformation with a Mg2+ ion coordinated by a conserved π-helix. In the absence of Mg2+-ATP, the CBS-pair domain adopts an elongated dimeric configuration with previously unobserved domain-domain contacts. Hydrogen-deuterium exchange mass spectrometry, analytical ultracentrifugation, and molecular dynamics experiments support a role of the structural rearrangements in mediating Mg2+-ATP sensing. Lastly, we use an in vitro, liposome-based assay to demonstrate direct Mg2+ transport by CorB proteins. These structural and functional insights provide a framework for understanding function of CNNMs in Mg2+ transport and associated diseases.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Hydrogenophilaceae/metabolismo , Magnésio/metabolismo , Methanomicrobiaceae/metabolismo , Sequência de Aminoácidos , Proteínas de Transporte de Cátions/genética , Cristalografia por Raios X , Medição da Troca de Deutério , Simulação de Dinâmica Molecular , Conformação Proteica , Domínios Proteicos
3.
Int J Syst Evol Microbiol ; 70(7): 4364-4371, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32579101

RESUMO

A novel, moderately thermophilic, Gram-stain-negative bacterium, designated strain J18T, was isolated from a water-flooded oil reservoir. Cells were aerobic, oxidase- and catalase-positive, with a polar flagellum. Growth occurred at 35-60 °C and at pH 6-8.5. The respiratory quinones were ubiquinone 8 and ubiquinone 9. The dominant cellular fatty acids were C16 : 0, C17 : 0 cyclo, C19 : 0 cyclo ω8c and summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c). The polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine, an unidentified aminolipid, an unidentified phospholipid and an unidentified aminophospholipid. The strain showed the highest 16S rRNA gene sequence similarities to Tepidiphilus margaritifer DSM 15129T (98.6 %), Tepidiphilus succinatimandens DSM 15512T (98.4 %) and Tepidiphilus thermophilus DSM 27220T (98.1 %), respectively, and the similarity to other species was lower than 93 %. In the phylogenetic trees, it constituted a unique sub-cluster within the genus Tepidiphilus. The DNA G+C content of strain J18T was 64.44 mol%. As compared with the type strains, the genome-to-genome distances of strain J18T were 34.7-40 %. These results confirmed the separate species status of J18T with its close relatives. On the basis of physiological, chemotaxonomic and phylogenetic analyses along with the low levels of identity at the whole-genome level, it can be concluded that strain J18T represents a new species of the genus Tepidiphilus, for which the name Tepidiphilus olei sp. nov. is proposed. The type strain of T. olei is J18T (=CGMCC 1.16800T=LMG 31400T).


Assuntos
Hydrogenophilaceae/classificação , Campos de Petróleo e Gás/microbiologia , Filogenia , Técnicas de Tipagem Bacteriana , Composição de Bases , China , DNA Bacteriano/genética , Ácidos Graxos/química , Hydrogenophilaceae/isolamento & purificação , Hibridização de Ácido Nucleico , Fosfolipídeos/química , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Ubiquinona/química , Água/análise
4.
Nat Commun ; 11(1): 2676, 2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-32472090

RESUMO

Cytochrome P450 monooxygenases are versatile heme-thiolate enzymes that catalyze a wide range of reactions. Self-sufficient cytochrome P450 enzymes contain the redox partners in a single polypeptide chain. Here, we present the crystal structure of full-length CYP116B46, a self-sufficient P450. The continuous polypeptide chain comprises three functional domains, which align well with the direction of electrons traveling from FMN to the heme through the [2Fe-2S] cluster. FMN and the [2Fe-2S] cluster are positioned closely, which facilitates efficient electron shuttling. The edge-to-edge straight-line distance between the [2Fe-2S] cluster and heme is approx. 25.3 Å. The role of several residues located between the [2Fe-2S] cluster and heme in the catalytic reaction is probed in mutagenesis experiments. These findings not only provide insights into the intramolecular electron transfer of self-sufficient P450s, but are also of interest for biotechnological applications of self-sufficient P450s.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Transporte de Elétrons/fisiologia , Hydrogenophilaceae/enzimologia , Cristalografia por Raios X , Conformação Proteica
5.
Curr Microbiol ; 77(8): 1939-1944, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32318862

RESUMO

A Gram-negative, aerobic, motile, non-spore-forming and rod-shaped bacterium, designated strain B18-69 T, was isolated from oil-well production liquid in Baolige oilfield, China. The strain was able to grow at pH 6-9.5 (optimum at pH 7), in 0-4% (w/v) NaCl (optimum at 0.5-1%, w/v) and at 35-60 °C (optimum at 55 °C). Major cellular fatty acids were C16:0, C19:0 cyclo ω8c, C17:0 cyclo and C18:1 ω7c. The predominant respiratory quinone was ubiquinone 8. Major polar lipids were phosphatidylethanolamine (PE), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG) and phosphatidylcholine (PC). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain B18-69 T was most closely related to Tepidiphilus margaritifer DSM 15129 T (98.8% similarity). The draft genome of strain B18-69 T was composed of 2,250,419 bp, and the G+C content was 64.6 mol%. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain B18-69 T and T. margaritifer DSM 15129 T were 90.9% and 68.9%, respectively. Genotypic and phenotypic features indicate that strain B18-69 T represents a novel species of the genus Tepidiphilus, for which the name Tepidiphilus baoligensis sp. nov. is proposed. The type strain is B18-69 T (= CGMCC 1.13573 T = KCTC 62782 T).


Assuntos
Hydrogenophilaceae/classificação , Campos de Petróleo e Gás/microbiologia , Filogenia , Técnicas de Tipagem Bacteriana , Composição de Bases , China , DNA Bacteriano/genética , Ácidos Graxos/química , Hydrogenophilaceae/isolamento & purificação , Hibridização de Ácido Nucleico , Fosfolipídeos/química , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Ubiquinona/química
6.
Appl Biochem Biotechnol ; 191(2): 881-892, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31907778

RESUMO

Enzymes used for adenosine triphosphate (ATP) synthesis play important roles in energy-dependent cascade reactions in vitro. In this study, two novel polyphosphate kinase 2 (PPK2) enzymes, HbPPK2 from Hydrogenophilaceae bacterium and NdPPK2 from Nocardioides dokdonensis, were characterized for ATP synthesis with the substrate polyphosphate (polyP). The optimum temperature and pH of both purified HbPPK2 and NdPPK2 were 30 °C and 6.5. HbPPK2 and NdPPK2 retained 30% and 14% of the initial activity at 30 °C for 12 h, respectively, whereas the presence of polyP significantly enhanced the stability of enzymes. The two PPK2s preferentially catalyzed the long-chain polyP hexametaphosphate as the phosphate donor. Adenosine monophosphate could not be used by HbPPK2 and NdPPK2 to synthesize ATP, indicating that they belonged to the class I subfamily of PPK2. HbPPK2 was used for ATP regeneration to produce glutathione by a two-enzyme cascade in vitro. 47.1 ± 0.4 mM glutathione was synthesized with a productivity of 13.5 ± 0.1 mM/h. ATP was regenerated approximately 471 times in the system within 3.5 h. HbPPK2 showed potential application for ATP regeneration in cascade reaction.


Assuntos
Trifosfato de Adenosina/biossíntese , Fosfotransferases (Aceptor do Grupo Fosfato)/química , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Monofosfato de Adenosina , Catálise , Estabilidade Enzimática , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Glutationa/metabolismo , Concentração de Íons de Hidrogênio , Hydrogenophilaceae/enzimologia , Nocardioides/enzimologia , Fosfatos , Fosfotransferases (Aceptor do Grupo Fosfato)/genética , Polifosfatos , Proteínas Recombinantes , Análise de Sequência , Especificidade por Substrato , Temperatura
7.
Antonie Van Leeuwenhoek ; 113(1): 83-99, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31531746

RESUMO

In the last decade there has been increased interest in the manipulation of rhizosphere microbial communities in soilless systems (hydroponics) through the addition of plant growth promoting microbes (PGPMs) to increase plant nutrition, lower plant stress response, and control pathogens. This method of crop management requires documenting patterns in communities living in plant roots throughout the growing season to inform decisions on timing of application and composition of the supplemental PGPM consortium. As a contribution to this effort, we measured changes in the bacterial community through early succession (first 26 days) in plant root biofilms growing in an indoor commercial aeroponic system where roots were sprayed with a mist of nutrient-amended water. By 12 days following seed germination, a root-associated community had established that was distinct from the source communities found circulating in the system. Successional patterns in the community over the following 2 weeks (12-26 days) included changes in abundance of bacterial groups that have been documented in published literature as able to utilize plant root exudates, release plant hormones, or augment nutrient availability. Six bacterial families/genera (Hydrogenophilaceae, Rhizobium, Legionellaceae, Methylophilus, Massilia, or Herbaspirillum) were the most abundant in each root sample, comprising 8-37% of the microbiome. Given the absence of soil-associated microbial communities in hydroponic systems, they provide an ideal design for isolating plant-microbial interactions and identifying key components possibly contributing to plant health.


Assuntos
Microbiota/genética , Rizosfera , Microbiologia do Solo , Herbaspirillum/classificação , Herbaspirillum/genética , Hydrogenophilaceae/classificação , Hydrogenophilaceae/genética , Legionellaceae/classificação , Legionellaceae/genética , Methylophilus/classificação , Methylophilus/genética , Rhizobium/classificação , Rhizobium/genética
8.
J Biosci Bioeng ; 127(6): 686-689, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30579830

RESUMO

Hydrogenophilus thermoluteolus strain TH-1 is a thermophilic hydrogen-oxidizing microorganism that has the highest growth rate among autotrophs. Genomic analysis revealed that this strain comprises the complete gene set for poly-ß-hydroxybutyrate (PHB) synthesis, i.e., three copies of acetyl-CoA acetyltransferase and polyhydroxyalkanoate synthase and one copy of acetoacetyl-CoA reductase and 3-hydroxyacyl-CoA dehydrogenase/3-hydroxybutyryl-CoA epimerase. An investigation on PHB accumulation in strain TH-1 demonstrated that PHB accumulation was induced by nitrogen limitation under autotrophic as well as heterotrophic conditions. This strain accumulated up to 430.4 ± 14.3 mg L-1 PHB during a 3-h incubation under nitrogen-limited heterotrophic conditions. The highest PHB accumulation rates under autotrophic and heterotrophic conditions were 38.6% (w/w) of the dry cells after a 6-h induction and 53.8% after 3 h, respectively. Although PHB granules started to accumulate after 15 min of nitrogen limitation under heterotrophic conditions, a drastic decrease of PHB was observed after 9 h of induction.


Assuntos
Hidrogênio/metabolismo , Hydrogenophilaceae/metabolismo , Hidroxibutiratos/metabolismo , Poliésteres/metabolismo , Temperatura , Aciltransferases/metabolismo , Oxirredutases do Álcool/metabolismo , Oxirredução , Oxirredutases/metabolismo
9.
Biochim Biophys Acta Bioenerg ; 1859(1): 8-18, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28970007

RESUMO

Biocatalysts that mediate the H2-dependent reduction of NAD+ to NADH are attractive from both a fundamental and applied perspective. Here we present the first biochemical and spectroscopic characterization of an NAD+-reducing [NiFe]­hydrogenase that sustains catalytic activity at high temperatures and in the presence of O2, which usually acts as an inhibitor. We isolated and sequenced the four structural genes, hoxFUYH, encoding the soluble NAD+-reducing [NiFe]­hydrogenase (SH) from the thermophilic betaproteobacterium, Hydrogenophilus thermoluteolus TH-1T (Ht). The HtSH was recombinantly overproduced in a hydrogenase-free mutant of the well-studied, H2-oxidizing betaproteobacterium Ralstonia eutropha H16 (Re). The enzyme was purified and characterized with various biochemical and spectroscopic techniques. Highest H2-mediated NAD+ reduction activity was observed at 80°C and pH6.5, and catalytic activity was found to be sustained at low O2 concentrations. Infrared spectroscopic analyses revealed a spectral pattern for as-isolated HtSH that is remarkably different from those of the closely related ReSH and other [NiFe]­hydrogenases. This indicates an unusual configuration of the oxidized catalytic center in HtSH. Complementary electron paramagnetic resonance spectroscopic analyses revealed spectral signatures similar to related NAD+-reducing [NiFe]­hydrogenases. This study lays the groundwork for structural and functional analyses of the HtSH as well as application of this enzyme for H2-driven cofactor recycling under oxic conditions at elevated temperatures.


Assuntos
Proteínas de Bactérias/química , Cupriavidus necator/enzimologia , Temperatura Alta , Hidrogênio/química , Hidrogenase/química , Hydrogenophilaceae/enzimologia , NAD/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cupriavidus necator/genética , Estabilidade Enzimática , Hidrogênio/metabolismo , Hidrogenase/genética , Hidrogenase/metabolismo , Hydrogenophilaceae/genética , NAD/metabolismo
10.
Int J Biol Macromol ; 106: 338-350, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28827133

RESUMO

An extracellular acido-thermostable endo-chitinase (called ChiA-Hh59) from thermophilic Hydrogenophilus hirschii strain KB-DZ44, was purified and characterized. The maximum chitinase activity recorded after 36-h of incubation at 60°C was 3000U/ml. Pure enzyme was obtained after heat and acidic treatment, precipitation by ammonium sulphate and acetone, respectively, followed by sequential column chromatographies on Sephacryl S-200 and Mono Q-Sepharose. Based on Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis, the purified enzyme is a monomer with a molecular mass of 59103.12-Da. The 22 residue NH2-terminal sequence of the enzyme showed high homology with family-18 bacterial chitinases. The optimum pH and temperature values for chitinase activity were pH 5.0 and 85°C, respectively. The pure enzyme was completely inhibited by p-chloromercuribenzoic acid (p-CMB) and N-ethylmaleimide (NEM). The obtained results suggest that ChiA-Hh59 might be an endo-chitinase. The studied chitinase exhibited high activity towards colloidal chitin, chitin azure, glycol chitin, while it did not hydrolyse chitibiose and amylose. Its Km and kcat values were 0.298mg colloidal chitin/ml and 14400s-1, respectively. Its catalytic efficiency was higher than those of chitodextrinase and ChiA-65. Additionally, Thin-layer chromatography (TLC) analysis from chitin-oligosaccharides showed that ChiA-Hh59 acted as an endo-splitting enzyme. In conclusion, this chitinase may have great potential for the enzymatic degradation of chitin.


Assuntos
Proteínas de Bactérias/química , Quitina/química , Quitinases/química , Hydrogenophilaceae/enzimologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/isolamento & purificação , Biocatálise , Quitinases/antagonistas & inibidores , Quitinases/isolamento & purificação , Inibidores Enzimáticos/química , Estabilidade Enzimática , Etilmaleimida/química , Expressão Gênica , Temperatura Alta , Concentração de Íons de Hidrogênio , Hydrogenophilaceae/química , Hydrogenophilaceae/classificação , Hidrólise , Cinética , Peso Molecular , Filogenia , Especificidade por Substrato , Ácido p-Cloromercurobenzoico/química
11.
Protein Sci ; 26(4): 737-748, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28097774

RESUMO

Thermophilic Hydrogenophilus thermoluteolus cytochrome c' (PHCP) exhibits higher thermal stability than a mesophilic counterpart, Allochromatium vinosum cytochrome c' (AVCP), which has a homo-dimeric structure and ligand-binding ability. To understand the thermal stability mechanism and ligand-binding ability of the thermally stable PHCP protein, the crystal structure of PHCP was first determined. It formed a homo-dimeric structure, the main chain root mean square deviation (rmsd) value between PHCP and AVCP being 0.65 Å. In the PHCP structure, six specific residues appeared to strengthen the heme-related and subunit-subunit interactions, which were not conserved in the AVCP structure. PHCP variants having altered subunit-subunit interactions were more severely destabilized than ones having altered heme-related interactions. The PHCP structure further revealed a ligand-binding channel and a penta-coordinated heme, as observed in the AVCP protein. A spectroscopic study clearly showed that some ligands were bound to the PHCP protein. It is concluded that the dimeric PHCP from the thermophile is effectively stabilized through heme-related and subunit-subunit interactions with conservation of the ligand-binding ability. BRIEF SUMMARY: We report the X-ray crystal structure of cytochrome c' (PHCP) from thermophilic Hydrogenophilus thermoluteolus. The high thermal stability of PHCP was attributed to heme-related and subunit-subunit interactions, which were confirmed by a mutagenesis study. The ligand-binding ability of PHCP was examined by spectrophotometry. PHCP acquired the thermal stability with conservation of the ligand-binding ability. This study furthers the understanding of the stability and function of cytochromes c.


Assuntos
Proteínas de Bactérias/química , Citocromos c'/química , Hydrogenophilaceae/enzimologia , Multimerização Proteica , Chromatiaceae/enzimologia , Cristalografia por Raios X , Estabilidade Enzimática , Temperatura Alta , Estrutura Quaternária de Proteína
12.
Biosci Biotechnol Biochem ; 79(7): 1125-9, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25752188

RESUMO

Cytochrome c' (SACP) from mesophilic Shewanella amazonensis, growing optimally at 37 °C, was thermally more stable than cytochrome c' (AVCP) from mesophilic Allochromatium vinosum, growing optimally at 25 °C. In contrast, SACP was less stable than cytochrome c' (PHCP) from thermophilic Hydrogenophilus thermoluteolus, growing optimally at 52 °C. Although only 28% of the SACP amino acid sequence was identical to those of AVCP and PHCP, the latter two being 55% identical, the overall main chain structures of the three cytochromes c' were similar, and SACP exhibited thermal stability intermediate between those of AVCP and PHCP. For these three proteins, the higher the stability is, the lesser the number of Gly residues in the putative α-helical regions is. Cytochromes c' including the present three are suitable for examining the protein stabilization mechanisms, because they are structurally similar and available from environments with a wide range of temperatures.


Assuntos
Citocromos c/química , Shewanella/enzimologia , Sequência de Aminoácidos , Chromatiaceae/enzimologia , Dicroísmo Circular , Citocromos c/metabolismo , Estabilidade Enzimática , Hydrogenophilaceae/enzimologia , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Desnaturação Proteica , Homologia de Sequência de Aminoácidos , Shewanella/crescimento & desenvolvimento , Temperatura , Termodinâmica
13.
Acta Crystallogr F Struct Biol Commun ; 71(Pt 1): 96-9, 2015 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-25615977

RESUMO

NAD+-reducing [NiFe] hydrogenases catalyze the oxidoreduction of dihydrogen concomitant with the interconversion of NAD+ and NADH. Here, the isolation, purification and crystallization of the NAD+-reducing [NiFe] hydrogenase from Hydrogenophilus thermoluteolus TH-1 are reported. Crystals of the NAD+-reducing [NiFe] hydrogenase were obtained within one week from a solution containing polyethylene glycol using the sitting-drop vapour-diffusion method and micro-seeding. The crystal diffracted to 2.58 Šresolution and belonged to space group C2, with unit-cell parameters a=131.43, b=189.71, c=124.59 Å, ß=109.42°. Assuming the presence of two NAD+-reducing [NiFe] hydrogenase molecules in the asymmetric unit, VM was calculated to be 2.2 Å3 Da(-1), which corresponds to a solvent content of 43%. Initial phases were determined by the single-wavelength anomalous dispersion method using the anomalous signal from the Fe atoms.


Assuntos
Proteínas de Bactérias/química , Hidrogenase/química , Hydrogenophilaceae/enzimologia , Cristalização , Cristalografia por Raios X
14.
Biosci Biotechnol Biochem ; 78(7): 1191-4, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25229856

RESUMO

Apo-cytochomes c without heme are usually unstructured. Here we showed that apo-form of thermophilic Hydrogenophilus thermoluteolus cytochrome c' (PHCP) was a monomeric protein with high helix content. Apo-PHCP was thermally stable, possibly due to the hydrophobic residues and ion pairs. PHCP is the first example of a structured apo-cytochrome c', which will expand our view of hemoprotein structure formation.


Assuntos
Citocromos c/química , Hydrogenophilaceae/enzimologia , Sequência de Aminoácidos , Apoenzimas/química , Estabilidade Enzimática , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína
15.
Biosci Biotechnol Biochem ; 77(8): 1677-81, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23924718

RESUMO

Sequence analysis indicated that thermophilic Hydrogenophilus thermoluteolus cytochrome c' (PHCP) and its mesophilic homolog, Allochromatium vinosum cytochrome c' (AVCP), closely resemble each other in a phylogenetic tree of the cytochrome c' family, with 55% sequence identity. The denaturation temperature of PHCP was 87 °C, 35 °C higher than that of AVCP. Furthermore, PHCP exhibited a larger enthalpy change value during its thermal denaturation than AVCP. While AVCP was dimeric, as observed previously, PHCP was trimeric, and this was the first observation as a cytochrome c'. Dissociation of trimeric PHCP and its protein denaturation reversibly occurred at the same time in a two-state transition manner. Therefore, PHCP is enthalpically more stable than AVCP, perhaps due to its unique trimeric form, in addition to the lower number of Gly residues in its putative α-helical regions.


Assuntos
Chromatiaceae/enzimologia , Citocromos c'/química , Estabilidade Enzimática , Hydrogenophilaceae/enzimologia , Sequência de Aminoácidos , Temperatura Alta , Filogenia , Desnaturação Proteica , Termodinâmica
16.
Environ Microbiol Rep ; 5(2): 235-42, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23584967

RESUMO

Hydrogenophilus is a thermophilic, facultative chemoautotroph, which lives prevalently in high temperature geothermal niches. Despite the environmental distribution, little is known about its oxidative phosphorylation. Here, we show that inverted membrane vesicles derived from Hydrogenophilus thermoluteolus cells autotrophically cultivated with H2 formed a proton gradient on the addition of succinate, dl-lactate, and NADH, and exhibited oxidation activity toward these three organic compounds. These indicate the capability of mixotrophic growth of this bacterium. Biochemical analysis demonstrated that the same vesicles contained an F-type ATP synthase. The F1 sector of the ATP synthase purified from H. thermoluteolus membranes exhibited optimal ATPase activity at 65°C. Transformed Escherichia coli membranes expressing H. thermoluteolus F-type ATP synthase exhibited the same temperature optimum for the ATPase. These findings shed light on H. thermoluteolus oxidative phosphorylation from the aspects of membrane bioenergetics and ATPase biochemistry, which must be fundamental and advantageous in the biogeochemical cycles occurred in the high temperature geothermal niches.


Assuntos
Hydrogenophilaceae/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular/química , Membrana Celular/enzimologia , Crescimento Quimioautotrófico , Ecossistema , Temperatura Alta , Hidrogênio/metabolismo , Hydrogenophilaceae/química , Hydrogenophilaceae/enzimologia , Hydrogenophilaceae/genética , Fosforilação Oxidativa
17.
Appl Environ Microbiol ; 78(18): 6545-9, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22773644

RESUMO

Except for several conspicuous cases, very little is known about sulfur oxidizers living in natural freshwater environments. Sulfuricella denitrificans skB26 is a psychrotolerant sulfur oxidizer recently isolated from a freshwater lake as a representative of a new genus in the class Betaproteobacteria. In this study, an approximately 3.2-Mb draft genome sequence of strain skB26 was obtained. In the draft genome, consisting of 23 contigs, a single rRNA operon, 43 tRNA genes, and 3,133 coding sequences were identified. The identified genes include those required for sulfur oxidation, denitrification, and carbon fixation. Comparative proteomic analysis was conducted to assess cold adaptation mechanisms of this organism. From cells grown at 22°C and 5°C, proteins were extracted for analysis by nano-liquid chromatography-electrospray ionization-tandem mass spectrometry. In the cells cultured at 5°C, relative abundances of ribosomal proteins, cold shock proteins, and DEAD/DEAH box RNA helicases were increased in comparison to those at 22°C. These results suggest that maintenance of proper translation is critical for growth under low-temperature conditions, similar to the case for other cold-adapted prokaryotes.


Assuntos
DNA Bacteriano/química , DNA Bacteriano/genética , Genoma Bacteriano , Hydrogenophilaceae/genética , Análise de Sequência de DNA , Proteínas de Bactérias/análise , Ciclo do Carbono , Temperatura Baixa , Desnitrificação , Água Doce/microbiologia , Hydrogenophilaceae/isolamento & purificação , Hydrogenophilaceae/fisiologia , Hydrogenophilaceae/efeitos da radiação , Lagos , Redes e Vias Metabólicas/genética , Dados de Sequência Molecular , Oxirredução , Proteoma/análise , RNA de Transferência/genética , Estresse Fisiológico , Compostos de Enxofre/metabolismo , Óperon de RNAr
18.
FEBS J ; 278(13): 2341-8, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21554540

RESUMO

Hydrogenophilus thermoluteolus cytochrome c' (PHCP) has typical spectral properties previously observed for other cytochromes c', which comprise Ambler's class II cytochromes c. The PHCP protein sequence (135 amino acids) deduced from the cloned gene is the most homologous (55% identity) to that of cytochrome c' from Allochromatium vinosum (AVCP). These findings indicate that PHCP forms a four-helix bundle structure, similar to AVCP. Strikingly, PHCP with a covalently bound heme was heterologously synthesized in the periplasm of Escherichia coli strains deficient in the DsbD protein, a component of the System I cytochrome c biogenesis machinery. The heterologous synthesis of PHCP by aerobically growing E. coli also occurred without a plasmid carrying the genes for Ccm proteins, other components of the System I machinery. Unlike Ambler's class I general cytochromes c, the synthesis of PHCP is not dependent on the System I machinery and exhibits similarity to that of E. coli periplasmic cytochrome b(562), a 106-residue four-helix bundle.


Assuntos
Chromatiaceae/metabolismo , Citocromos c'/metabolismo , Citocromos c/metabolismo , Escherichia coli/metabolismo , Heme/metabolismo , Hydrogenophilaceae/metabolismo , Periplasma/metabolismo , Sequência de Aminoácidos , Chromatiaceae/genética , Citocromos c/genética , Citocromos c/isolamento & purificação , Citocromos c'/genética , Citocromos c'/isolamento & purificação , Escherichia coli/genética , Hydrogenophilaceae/genética , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos
19.
Int J Syst Evol Microbiol ; 61(Pt 2): 290-294, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20228213

RESUMO

A novel chemolithotrophic bacterium, strain 16C(T), was isolated from a hot spring in Graendalur, south-west Iceland. Cells of this organism were Gram-negative, rod-shaped and motile. The isolate was aerobic and capable of chemolithotrophic growth on hydrogen and carbon dioxide, heterotrophic growth on butyrate and several other organic compounds, and mixotrophic growth on butyrate, hydrogen and carbon dioxide. Heterotrophic growth was generally enhanced in the presence of yeast extract. Autotrophic growth on hydrogen was observed at pH values between 6.0 and 10.0 and temperatures between 35 and 60 °C; optimum growth conditions were pH 7.0 and 55 °C. The DNA G+C content was 63.9 mol%. 16S rRNA gene sequence analysis showed that strain 16C(T) was a member of a distinct species belonging to the class Betaproteobacteria and was most closely related to Hydrogenophilus thermoluteolus NBRC 14978(T) and Hydrogenophilus hirschii DSM 11420(T). The major cellular fatty acids were straight-chain C(16 : 0) (44.98 %) and C(18 : 1)ω7c (17.93 %), as well as cyclic C(17 : 0) (13.90 %) and C(19 : 0)ω8c (4.67 %) fatty acids. Based on its physiological and molecular properties, it is concluded that strain 16C(T) represents a novel species within the genus Hydrogenophilus, for which the name Hydrogenophilus islandicus is proposed; the type strain is 16C(T) (=DSM 21442(T)=JCM 16106(T)).


Assuntos
Fontes Termais/microbiologia , Hydrogenophilaceae/classificação , Filogenia , Microbiologia da Água , Técnicas de Tipagem Bacteriana , Composição de Bases , Dióxido de Carbono/metabolismo , DNA Bacteriano/genética , Ácidos Graxos/análise , Processos Heterotróficos , Temperatura Alta , Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Hydrogenophilaceae/genética , Hydrogenophilaceae/isolamento & purificação , Islândia , Dados de Sequência Molecular , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
20.
Microbiology (Reading) ; 155(Pt 4): 1302-1309, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19332831

RESUMO

The halotolerant acidophile 'Thiobacillus prosperus' was shown to require chloride for growth. With ferrous iron as substrate, growth occurred at a rate similar to that of the well-studied acidophile Acidithiobacillus ferrooxidans. Previously, the salt (NaCl) requirement of 'T. prosperus' was not clear and its growth on ferrous iron was described as poor. A subtractive hybridization of cDNAs from ferrous-iron-grown and sulfur-grown 'T. prosperus' strain V6 led to identification of a cluster of genes similar to the rus operon reported to encode ferrous iron oxidation in A. ferrooxidans. However, the 'T. prosperus' gene cluster did not contain a homologue of cyc1, which is thought to encode a key cytochrome c in the pathway of electron transport from ferrous iron in A. ferrooxidans. Rusticyanin, another key protein in ferrous iron oxidation by A. ferrooxidans, was present in 'T. prosperus' at similar concentrations in cells grown on either ferrous iron or sulfur.


Assuntos
Azurina/metabolismo , Compostos Ferrosos/metabolismo , Hydrogenophilaceae/metabolismo , Ferro/metabolismo , Cloreto de Sódio , Enxofre , Sequência de Aminoácidos , Azurina/química , Azurina/genética , DNA Bacteriano/análise , Concentração de Íons de Hidrogênio , Hydrogenophilaceae/genética , Hydrogenophilaceae/crescimento & desenvolvimento , Hydrogenophilaceae/fisiologia , Dados de Sequência Molecular , Família Multigênica , Oxirredução , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Cloreto de Sódio/metabolismo , Enxofre/metabolismo
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