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1.
Int J Syst Evol Microbiol ; 70(10): 5503-5511, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32910752

RESUMO

Two Gram-stain-positive, strictly aerobic, non-spore-forming actinobacterial strains, designated YC2-7T and YC5-17, were isolated from the Yongcheondonggul (larva cave) in Jeju, Republic of Korea and their taxonomic ranks were examined by a polyphasic approach. The 16S rRNA gene tree showed that the novel isolates occupied an independent position separated from recognized genera of the family Nocardiaceae. In the 92 core gene-based phylogenomic analysis, strain YC2-7T was loosely associated with the type strain of Aldersonia kummingensis with 66.2 % average amino acid identity. The 16S rRNA gene sequence simairity between the isolate and members of the family Nocardiaceae was below 96.7 %. The cell-wall peptidoglycan was meso-diaminopimelic acid as a diagnostic diamino acid. Whole-cell sugars consisted of arabinose, galactose and glucose. The predominant menaquinone was MK-8(H4, ω-cycl). The major polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannoside. The cellular fatty acids consisted mainly of saturated and unsaturated components with small amounts of tuberculostearic acid. Mycolic acids of 52-58 carbon atoms were present. The DNA G+C content of the genome was 63.8 mol%. On the basis of combination of morphological and chemotaxonomic differences, in addition to phylogenetic distinctness, the novel isolates are considered to constitute members of a novel species of a new genus in the family Nocardiaceae, for which the name Antrihabitans stalactiti gen. nov., sp. nov. is proposed. The type strain is YC2-7T (=KACC 19965T=DSM 108733T).


Assuntos
Cavernas/microbiologia , Nocardiaceae/classificação , Filogenia , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácido Diaminopimélico/química , Ácidos Graxos/química , Ácidos Micólicos/química , Nocardiaceae/isolamento & purificação , Peptidoglicano/química , Fosfolipídeos/química , RNA Ribossômico 16S/genética , República da Coreia , Análise de Sequência de DNA , Vitamina K 2/análogos & derivados , Vitamina K 2/química
2.
Int. microbiol ; 22(4): 521-530, dic. 2019. mapas, graf, tab
Artigo em Inglês | IBECS | ID: ibc-185070

RESUMO

A total of 168 actinomycete colonies were isolated from 14 sediment samples of the northern parts of the Oman Sea and were screened for cytotoxic and antimicrobial activity. Among four media and two treatments, the glucose arginine agar medium (18%) and heat treatment (28.3%) showed maximum isolation rate of actinomycetes. Preliminary characterization revealed that the members of Streptomycetaceae were widely distributed (66%) in the most of the sampling stations followed by Micromonosporaceae (14%), Nocardiaceae (6%), and Pseudonocardiaceae (4%), respectively. Approximately, 23.8% of the isolates inhibited the growth of at least one of the microbial test strains, while the majority of them belonged to the Streptomycetaceae family. Minimum inhibitory concentrations of the ethyl acetate culture extracts of the five most putative isolates varied from 64 μg/mL against Micrococcus luteus and Candida albicans to 1 mg/mL against Aspergillus niger. These extracts showed significant cytotoxic activity at18.74-193.5 μg/mL on the human breast (MCF7), colon (HCT 116), and liver (HepG2) tumor cell lines while exhibited less or no cytotoxicity on the normal cell line (HUVEC). Interestingly, IFSRI 193 extract selectively inhibited the growth of HCT 116 cell line and gram-positive bacteria. 16S rRNA gene sequencing revealed that the potent isolates have 97 to 99% similarity with S. chartreusis, S. cacaoi, S. sampsonii, S. qinglanensis, and S. diastaticus. These results suggested that the five Streptomyces strains could be considered candidates for discovering the antitumor antibiotics


No disponible


Assuntos
Infecções por Actinomycetales/microbiologia , Sedimentos Marinhos (Saúde Ambiental)/análise , Streptomycetaceae/isolamento & purificação , Micromonosporaceae/isolamento & purificação , Nocardiaceae/isolamento & purificação , RNA Ribossômico 16S/isolamento & purificação , Células HCT116/metabolismo , Células HCT116/microbiologia
3.
J Hazard Mater ; 378: 120618, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31301927

RESUMO

This study employed innovative technologies to evaluate multiple lines of evidence for natural attenuation (NA) of methyl tertiary-butyl ether (MTBE) in groundwater at the 22 Area of Marine Corps Base (MCB) Camp Pendleton after decommissioning of a biobarrier system. For comparison, data from the 13 Area Gas Station where active treatment of MTBE is occurring was used to evaluate the effectiveness of omic techniques in assessing biodegradation. Overall, the 22 Area Gas Station appeared to be anoxic. MTBE was detected in large portion of the plume. In comparison, concentrations of MTBE at the 13 Area Gas Station were much higher (42,000 µg/L to 2800 µg/L); however, none of the oxygenates were detected. Metagenomic analysis of the indigenous groundwater microbial community revealed the presence of bacterial strains known to aerobically and anaerobically degrade MTBE at both sites. While proteomic analysis at the 22 Area Gas Station showed the presence of proteins of MTBE degrading microorganisms, the MTBE degradative proteins were only found at the 13 Area Gas Station. Taken together, these results provide evidence for previous NA of MTBE in the groundwater at 22 Area Gas Station and demonstrate the effectiveness of innovative-omic technologies to assist monitored NA assessments.


Assuntos
Biodegradação Ambiental , Genômica , Água Subterrânea/química , Éteres Metílicos/química , Proteômica , Poluentes Químicos da Água/química , California , Monitoramento Ambiental/métodos , Hidrocarbonetos/química , Nocardiaceae/metabolismo , Peptídeos/química , Petróleo , Pseudomonas/metabolismo , terc-Butil Álcool
4.
Mol Ecol ; 28(4): 900-916, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30106217

RESUMO

Acacia-ant mutualists in the genus Pseudomyrmex nest obligately in acacia plants and, as we show through stable isotope analysis, feed at a remarkably low trophic level. Insects with diets such as these sometimes depend on bacterial symbionts for nutritional enrichment. We, therefore, examine the bacterial communities associated with acacia-ants in order to determine whether they host bacterial partners likely to contribute to their nutrition. Despite large differences in trophic position, acacia-ants and related species with generalized diets do not host distinct bacterial taxa. However, we find that a small number of previously undescribed bacterial taxa do differ in relative abundance between acacia-ants and generalists, including several Acetobacteraceae and Nocardiaceae lineages related to common insect associates. Comparisons with an herbivorous generalist, a parasite that feeds on acacias and a mutualistic species with a generalized diet show that trophic level is likely responsible for these small differences in bacterial community structure. While we did not experimentally test for a nutritional benefit to hosts of these bacterial lineages, metagenomic analysis reveals a Bartonella relative with an intact nitrogen-recycling pathway widespread across Pseudomyrmex mutualists and generalists. This taxon may be contributing to nitrogen enrichment of its ant hosts through urease activity and, concordant with an obligately host-associated lifestyle, appears to be experiencing genomewide relaxed selection. The lack of distinctiveness in bacterial communities across trophic level in this group of ants shows a remarkable ability to adjust to varied diets, possibly with assistance from these diverse ant-specific bacterial lineages.


Assuntos
Acacia/fisiologia , Formigas/fisiologia , Acetobacteraceae/fisiologia , Animais , Bartonella/fisiologia , Herbivoria/fisiologia , Metagenômica , Microbiota/fisiologia , Nocardiaceae/fisiologia , Simbiose/genética , Simbiose/fisiologia
6.
J Gen Appl Microbiol ; 64(2): 62-67, 2018 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-29367492

RESUMO

A new antibiotic, designated mumiamicin, was isolated from the cultured broth of the rare actinomycete strain, Mumia sp. YSP-2-79, by Diaion HP-20, silica gel and ODS column chromatography, followed by HPLC purification. The chemical structure of mumiamicin was elucidated as a new furan fatty acid by nuclear magnetic resonance and mass spectrometry. Mumiamicin showed antimicrobial activity and antioxidative activity.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Ácidos Graxos/química , Furanos/química , Furanos/farmacologia , Nocardiaceae/química , Animais , Antibacterianos/isolamento & purificação , Antioxidantes/química , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Cromatografia Líquida de Alta Pressão , Ácidos Graxos/isolamento & purificação , Ácidos Graxos/farmacologia , Fermentação , Furanos/isolamento & purificação , Espectroscopia de Ressonância Magnética , Nocardiaceae/classificação , Nocardiaceae/isolamento & purificação , Poríferos/microbiologia
7.
Fish Shellfish Immunol ; 55: 281-92, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27238429

RESUMO

Interleukin (IL) -12 is a heterodimeric cytokine mainly produced by monocytes, macrophages, and dendritic cells in mammals. IL-12p70 composed of IL-12p35 and IL-12p40, is known to play a crucial role in promoting cell-mediated immunity (CMI) through Th1 differentiation and IFN-γ production. Although two types of IL-12p35 (p35a, p35b) and three types of IL-12p40 (p40a, p40b and p40c) have been identified in several fish species, the knowledge on functional characteristics of teleost IL-12 is still limited. In the present study, we cloned two types of IL-12p35 and three types of IL-12p40 genes in amberjack and yellowtail, and analyzed their expressions in response to stimulation with Nocardia seriolae in amberjack. As a result, four types of IL-12 (IL-12p35a, p35b, p40a and p40b) and IFN-γ mRNA were increased by live-N. seriolae stimulation but not by formalin-killed N. seriolae, suggesting that four types of IL-12 (p35, p35b, p40a and p40c) participate in promoting CMI. Subsequently, we produced six types of recombinant IL-12p70 (rIL12p70) protein in insect cells. Head kidney leukocytes were cultured with formalin-killed N. seriolae and six types of rIL-12p70 to elucidate the role of amberjack IL-12p70 in induction of CMI. After stimulation, IFN-γ expression was elevated whereas IL-10 expression was suppressed in Head kidney leukocytes stimulated with four types of rIL-12 (p40a/p35a, p40c/p35a, p40a/p35b, p40a/p35b). On the other hand, two types of rIL-12 (p40b/p35a, p40b/p35b) only elicited down regulation of IL-10 expression. These results indicate that all amberjack IL-12p70 isoforms are involved in Th1 -differentiation and promotion of CMI with different manners. Fish IL-12 has a potential for the promising vaccine adjuvant.


Assuntos
Infecções por Actinomycetales/veterinária , Vacinas Bacterianas/imunologia , Doenças dos Peixes/terapia , Proteínas de Peixes/genética , Interleucina-12/genética , Nocardiaceae/imunologia , Perciformes , Infecções por Actinomycetales/microbiologia , Infecções por Actinomycetales/terapia , Sequência de Aminoácidos , Animais , Doenças dos Peixes/microbiologia , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Interleucina-12/química , Interleucina-12/metabolismo , Subunidade p35 da Interleucina-12/genética , Subunidade p35 da Interleucina-12/metabolismo , Subunidade p40 da Interleucina-12/genética , Subunidade p40 da Interleucina-12/metabolismo , Filogenia , Alinhamento de Sequência/veterinária , Vacinas Atenuadas/imunologia , Vacinas de Produtos Inativados/imunologia , Vacinas Sintéticas/imunologia
8.
Microbiome ; 4: 3, 2016 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-26792212

RESUMO

BACKGROUND: While 16S ribosomal RNA (rRNA) sequencing has been used to characterize the lung's bacterial microbiota in human immunodeficiency virus (HIV)-infected individuals, taxonomic studies provide limited information on bacterial function and impact on the host. Metabolic profiles can provide functional information on host-microbe interactions in the lungs. We investigated the relationship between the respiratory microbiota and metabolic profiles in the bronchoalveolar lavage fluid of HIV-infected and HIV-uninfected outpatients. RESULTS: Targeted sequencing of the 16S rRNA gene was used to analyze the bacterial community structure and liquid chromatography-high-resolution mass spectrometry was used to detect features in bronchoalveolar lavage fluid. Global integration of all metabolic features with microbial species was done using sparse partial least squares regression. Thirty-nine HIV-infected subjects and 20 HIV-uninfected controls without acute respiratory symptoms were enrolled. Twelve mass-to-charge ratio (m/z) features from C18 analysis were significantly different between HIV-infected individuals and controls (false discovery rate (FDR) = 0.2); another 79 features were identified by network analysis. Further metabolite analysis demonstrated that four features were significantly overrepresented in the bronchoalveolar lavage (BAL) fluid of HIV-infected individuals compared to HIV-uninfected, including cystine, two complex carbohydrates, and 3,5-dibromo-L-tyrosine. There were 231 m/z features significantly associated with peripheral blood CD4 cell counts identified using sparse partial least squares regression (sPLS) at a variable importance on projection (VIP) threshold of 2. Twenty-five percent of these 91 m/z features were associated with various microbial species. Bacteria from families Caulobacteraceae, Staphylococcaceae, Nocardioidaceae, and genus Streptococcus were associated with the greatest number of features. Glycerophospholipid and lineolate pathways correlated with these bacteria. CONCLUSIONS: In bronchoalveolar lavage fluid, specific metabolic profiles correlated with bacterial organisms known to play a role in the pathogenesis of pneumonia in HIV-infected individuals. These findings suggest that microbial communities and their interactions with the host may have functional metabolic impact in the lung.


Assuntos
Infecções por HIV/metabolismo , Infecções por HIV/microbiologia , Pulmão/metabolismo , Metaboloma , Microbiota/genética , RNA Ribossômico 16S/genética , Adulto , Líquido da Lavagem Broncoalveolar/microbiologia , Estudos de Casos e Controles , Caulobacteraceae/classificação , Caulobacteraceae/genética , Caulobacteraceae/metabolismo , Cromatografia Líquida , Cistina/metabolismo , Feminino , Glicerofosfolipídeos/metabolismo , HIV/crescimento & desenvolvimento , Infecções por HIV/virologia , Interações Hospedeiro-Patógeno , Humanos , Análise dos Mínimos Quadrados , Pulmão/microbiologia , Masculino , Espectrometria de Massas , Nocardiaceae/classificação , Nocardiaceae/genética , Nocardiaceae/metabolismo , RNA Ribossômico 16S/metabolismo , Análise de Sequência de RNA , Staphylococcaceae/classificação , Staphylococcaceae/genética , Staphylococcaceae/metabolismo , Streptococcus/classificação , Streptococcus/genética , Streptococcus/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismo
9.
Chem Biodivers ; 11(8): 1151-62, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25146761

RESUMO

In this work, two protein systems, Kij3D-FMN-AKM-O2 and Kij3D-FMN-O2 , made of KijD3 N-oxygenase, flavin mononucleotide (FMN) cofactor, dTDP-3-amino-2,3,6-trideoxy-4-keto-3-methyl-D-glucose (AKM) substrate, and dioxygen (O2), have been assembled by adding a molecule of O2, and removing (or not) AKM, to crystal data for the Kij3D-FMN-AKM complex. Egress of AKM and O2 from these systems was then investigated by applying a tiny external random force, in turn, to their center of mass in the course of molecular dynamics in explicit H2 O. It turned out that the wide AKM channel, even when emptied, does not constitute the main route for O2 egress. Other routes appear to be also viable, while various binding pockets (BPs) outside the active center are prone to trap O2. By reversing the reasoning, these can also be considered as routes for uptake of O2 by the protein, before or after AKM uptake, while BPs may serve as reservoirs of O2. This shows that the small molecule O2 is capable of permeating the protein by exploiting all nearby interstices that are created on thermal fluctuations of the protein, rather than having necessarily to look for farther, permanent channels.


Assuntos
Mononucleotídeo de Flavina/química , Oxigênio/metabolismo , Oxigenases/química , Oxigenases/metabolismo , Sítios de Ligação , Mononucleotídeo de Flavina/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Nocardiaceae/enzimologia , Conformação Proteica
10.
Wei Sheng Wu Xue Bao ; 52(6): 671-8, 2012 Jun 04.
Artigo em Chinês | MEDLINE | ID: mdl-22934346

RESUMO

The Nocardioides genus was established by Prauser H. in 1976 according to morphological and physiological characteristics, as well as partial chemotaxonomic analyses of 17 nocardio-form actinobacteria isolated from soil, based on which a novel species Nocardioides albus was proposed as the type species. With the development in the technologies of isolation, purification and taxonomy, more and more members of this genus with varied morphological, physiological and biochemical characteristics were increasingly discovered from different sources, while all of them shared the same diagnostic characteristics of the genus. In the past 50 years, some of the members of the genus Nocardioides were ever transferred in or out and then some species description was ever emended. Till date, there were 56 validly described species in this genus. Some members of this genus were used in agriculture and industry. The objective of this review is to summarize the research advances in the genus Nocardioides, such as the changes of the taxonomic position and emendation description of the species as well as the application prospect in industry and agriculture.


Assuntos
Nocardiaceae/classificação , Nocardiaceae/genética , Filogenia
11.
J Biol Chem ; 285(33): 25782-91, 2010 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-20538584

RESUMO

Histamine dehydrogenase (HADH) isolated from Nocardioides simplex catalyzes the oxidative deamination of histamine to imidazole acetaldehyde. HADH is highly specific for histamine, and we are interested in understanding the recognition mode of histamine in its active site. We describe the first crystal structure of a recombinant form of HADH (HADH) to 2.7-A resolution. HADH is a homodimer, where each 76-kDa subunit contains an iron-sulfur cluster ([4Fe-4S](2+)) and a 6-S-cysteinyl flavin mononucleotide (6-S-Cys-FMN) as redox cofactors. The overall structure of HADH is very similar to that of trimethylamine dehydrogenase (TMADH) from Methylotrophus methylophilus (bacterium W3A1). However, some distinct differences between the structure of HADH and TMADH have been found. Tyr(60), Trp(264), and Trp(355) provide the framework for the "aromatic bowl" that serves as a trimethylamine-binding site in TMADH is comprised of Gln(65), Trp(267), and Asp(358), respectively, in HADH. The surface Tyr(442) that is essential in transferring electrons to electron-transfer flavoprotein (ETF) in TMADH is not conserved in HADH. We use this structure to propose the binding mode for histamine in the active site of HADH through molecular modeling and to compare the interactions to those observed for other histamine-binding proteins whose structures are known.


Assuntos
Nocardiaceae/enzimologia , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/química , Cristalografia por Raios X , Estrutura Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
12.
Biochemistry ; 49(17): 3517-24, 2010 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-20334431

RESUMO

D-kijanose is an unusual nitrosugar found attached to the antibiotic kijanimicin. Ten enzymes are required for its production in Actinomadura kijaniata, a soil-dwelling actinomycete. The focus of this investigation is on the protein encoded by the kijd3 gene and hereafter referred to as KijD3. On the basis of amino acid sequence analyses, KijD3 has been proposed to be an FAD-dependent oxidoreductase, which catalyzes the sixth step in d-kijanose biosynthesis by converting dTDP-3-amino-2,3,6-trideoxy-4-keto-3-methyl-d-glucose into its C-3' nitro derivative. This putative activity, however, has never been demonstrated in vivo or in vitro. Here we report the first structural study of this enzyme. For our investigation, crystals of KijD3 were grown in the presence of dTDP, and the structure was solved to 2.05-A resolution. The enzyme is a tetramer with each subunit folding into three distinct regions: a five alpha-helical bundle, an eight-stranded beta-sheet, and a second five alpha-helical bundle. The dTDP moiety is anchored to the protein via the side chains of Glu 113, Gln 254, and Arg 330. The overall fold of KijD3 places it into the well-characterized fatty acyl-CoA dehydrogenase superfamily. There is a decided cleft in each subunit with the appropriate dimensions to accommodate a dTDP-linked sugar. Strikingly, the loop defined by Phe 383 to Ala 388, which projects into the active site, contains two adjacent cis-peptide bonds, Pro 386 and Tyr 387. Activity assays demonstrate that KijD3 requires FAD for activity and that it produces a hydroxylamino product. The molecular architecture of KijD3 described in this report serves as a paradigm for a new family of enzymes that function on dTDP-linked sugar substrates.


Assuntos
Acil-CoA Desidrogenase/química , Acil-CoA Desidrogenase/metabolismo , Aminoglicosídeos/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Nitrorredutases/química , Nitrorredutases/metabolismo , Nocardiaceae/enzimologia , Acil-CoA Desidrogenase/genética , Catálise , Domínio Catalítico , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Dimerização , Modelos Moleculares , Nitrorredutases/genética , Conformação Proteica
13.
Colloids Surf B Biointerfaces ; 78(1): 8-16, 2010 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-20202801

RESUMO

Considering the need of potential biosurfactant producers and economic production processes using industrial waste, the present study aims to develop solid-state culture (SSC) of a marine actinobacterium for biosurfactant production. A potential biosurfactant producer Nocardiopsis lucentensis MSA04 was isolated from the marine sponge Dendrilla nigra. Among the substrates screened, wheat bran increased the production significantly (E(24) 25%) followed by oil seed cake and industrial waste such as tannery pretreated sludge, treated molasses (distillery waste) and pretreated molasses. Enhanced biosurfactant production was achieved under SSC conditions using kerosene as carbon source, beef extract as nitrogen source and wheat bran as substrate. The maximum production of biosurfactant by MSA04 occurred at a C/N ratio of 0.5 envisaging that a higher amount of nitrogen source is required by the strain compared to that of the carbon source. The kerosene and beef extract interactively increase the production and a stable production was attained with the influence of both factors independently. A significant interactive influence of secondary control factors such as copper sulfate and inoculum size was validated in response surface methods-based experiments. The surface active compound produced by MSA04 was characterized as glycolipid with a hydrophobic non-polar hydrocarbon chain (nonanoic acid methyl ester) and hydrophilic sugar, 3-acetyl 2,5 dimethyl furan. In conclusion, the strain N. lucentensis MSA04 was a potential source of glycolipid biosurfactant, could be used for the development of bioremediation processes in the marine environment.


Assuntos
Técnicas de Cultura de Células/métodos , Glicolipídeos/biossíntese , Nocardiaceae/química , Nocardiaceae/citologia , Água do Mar/microbiologia , Tensoativos/metabolismo , Análise de Variância , Carbono/farmacologia , Genes Bacterianos/genética , Glicolipídeos/química , Glicolipídeos/isolamento & purificação , Hidrocarbonetos/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Querosene , Espectrometria de Massas , Nitrogênio/farmacologia , Nocardiaceae/genética , Nocardiaceae/isolamento & purificação , Filogenia , Propriedades de Superfície/efeitos dos fármacos , Tensoativos/química , Tensoativos/isolamento & purificação
14.
J Biochem ; 147(2): 257-64, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19846434

RESUMO

Histamine dehydrogenase from Nocardioides simplex (nHmDH) is a homodimer containing one 6-S-cysteinyl FMN (CFMN) and one [4Fe-4S] cluster per monomer. nHmDH catalyses the oxidative deamination of histamine to ammonia and imidazole acetaldehyde, but histamine inhibits its catalytic activity at high concentrations. We mutated gene-encoded residues (Tyr180, Gly268 and Asp269) near CFMN to understand the biophysical meaning of the substrate inhibition. Three mutants Y180F, G268D/D269C and Y180F/G268D/D269C were expressed by considering the DNA sequence alignment of histamine dehydrogenase from Rhizobium sp. 4-9 (rHmDH), which does not suffer from the substrate inhibition. The Y180F/G268D/D269C mutation to mimic rHmDH successfully suppressed the inhibition, although the catalytic activity decreased. The substrate inhibition was weakened by the Y180F mutation, but G268D/D269C was still susceptible to the inhibition. It was found that it also causes changes in the UV-vis absorption spectra of the substrate-reduced form and the redox potential of the enzymes. The characterization suggests that the thermodynamic preference of the semiquinone form of CFMN in the two-electron-reduced subunit of the enzyme is responsible for the substrate inhibition. However, destabilization of the semiquinone form leads to kinetic hindrance due to the uphill single electron transfer from the fully reduced CFMN to the [4Fe-4S] cluster.


Assuntos
Mutagênese Sítio-Dirigida , Nocardiaceae/enzimologia , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Sequência de Aminoácidos , Domínio Catalítico/genética , Cristalografia por Raios X , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Cinética , Dados de Sequência Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/química , Homologia de Sequência de Aminoácidos , Termodinâmica
15.
Langmuir ; 25(18): 10660-5, 2009 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-19735135

RESUMO

We have synthesized a novel interface-anchoring alcohol dehydrogenase by covalent attachment of a hydrophobic polymer tail to the hydrophilic protein head. Analogous to a protein-based surfactant, this polymer-enzyme conjugate self-assembled at liquid-liquid or liquid-air interfaces to form a membrane similar to other surfactant monolayers. The packing and morphology of the interface-anchored enzymes play an important role in regulating the membrane behaviors including enzyme mobility and interfacial interactions of enzymes with reactant and product molecules. To characterize the surface assembly morphology of the interface-anchored enzymes, Langmuir film balance and fluorescence microscopy techniques were used. The Langmuir isotherm of the interface-anchored enzyme demonstrated a pronounced molecular rearrangement upon compression of the isotherm. This corresponded to changes in membrane morphology and state observed using fluorescence microscopy. The molecular diffusion within the novel interface-anchored enzymes was further evaluated by using a fluorescence recovery after photobleaching technique. We report a diffusion coefficient of 6.7x10(-10) cm2/s. The study represents the first in-depth analysis of surface packing and interfacial mobility of such interface-anchored enzymes.


Assuntos
Álcool Desidrogenase/química , Álcool Desidrogenase/metabolismo , Acetofenonas , Adsorção , Difusão , Recuperação de Fluorescência Após Fotodegradação , Interações Hidrofóbicas e Hidrofílicas , Microscopia de Fluorescência , Nocardiaceae/enzimologia , Polímeros/química , Pressão
16.
FEBS J ; 276(4): 1048-58, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19154353

RESUMO

Bacterial and archaeal endo-beta-1,3-glucanases that belong to glycoside hydrolase family 16 share a beta-jelly-roll fold, but differ significantly in sequence and in substrate specificity. The crystal structure of the laminarinase (EC 3.2.1.39) from the hyperthermophilic archaeon Pyrococcus furiosus (pfLamA) has been determined at 2.1 A resolution by molecular replacement. The pfLamA structure reveals a kink of six residues (72-77) at the entrance of the catalytic cleft. This peptide is absent in the endoglucanases from alkaliphilic Nocardiopsis sp. strain F96 and Bacillus macerans, two proteins displaying an overall fold similar to that of pfLamA, but with different substrate specificity. A deletion mutant of pfLamA, lacking residues 72-75, hydrolyses the mixed-linkage beta-1,3-1,4-glucan lichenan 10 times more efficiently than the wild-type protein, indicating the importance of the kink in substrate preference.


Assuntos
Proteínas de Bactérias/química , Celulases/química , Pyrococcus furiosus/enzimologia , Sequência de Aminoácidos , Bacillus/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cálcio/metabolismo , Catálise , Celulases/genética , Celulases/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Nocardiaceae/enzimologia , Conformação Proteica , Especificidade por Substrato
17.
Actual. SIDA ; 16(62): 145-149, nov. 2008. ilus
Artigo em Espanhol | LILACS | ID: lil-516529

RESUMO

Tsukamurella spp. es un bacilo gram positivo, aeróbico, catalasa positivo, no móvil, no esporulado, que pertenece al orden de los actinomicetales. Los géneros incluidos en este orden son Nocardia, Gordonia, dietza, Skermania, Williamsia, Turicella, Streptomyces y Rhodococcus. Otros géneros relacionados son Corynebacterium y Mycobacterium. Las infecciones por esos microorganismos se han asociado con neumopatías crónicas, inmunodepresión (leucemia, tumores, infección por el HIV) e infecciones postoperatorias de heridas. Se notificó la presencia de tsukamurella en hemocultivos asociada al uso de sondas o catéteres, otros dispositivos médicos y en casos individuales de tenosinovitis necrosante con abscesos subcutáneos, infecciones óseas y cutáneas, meningitis, peritonitis y conjuntivitis y también como germen colonizante. Se presenta un caso de otomastoiditis en un paciente HIV positivo causado por este germen.


Assuntos
Humanos , Masculino , Adulto , Terapia Antirretroviral de Alta Atividade , Diplopia/patologia , HIV , Mastoidite/terapia , Nocardiaceae/imunologia
18.
Biosci Biotechnol Biochem ; 72(3): 786-96, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18323648

RESUMO

Histamine dehydrogenase from Nocardioides simplex is a homodimer and belongs to the family of iron-sulfur flavoproteins having one [4Fe-4S] cluster and one 6-S-cysteinyl FMN per monomer. In the reductive titration with histamine, two-electron reduction occurred per monomer at pH<9, while single-electron reduction proceeded at pH>9. The substrate-reduced histamine dehydrogenase yielded an electron paramagnetic resonance spectral signal assigned to the flavin semiquinone. The signal intensity increased with pH up to pH 9 and reached a maximum at pH>9. These unique features are explained in terms of the redox potential of the cofactors, where the redox potential was evaluated over a pH range from 7 to 10 by using a spectroelectrochemical titration method for the flavin and cyclic voltammetry for the [4Fe-4S] cluster. The bell-type pH dependence of the enzymatic activity is also discussed in terms of the pH dependence of the centers' redox potential.


Assuntos
Nocardiaceae/enzimologia , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/química , Termodinâmica , Espectroscopia de Ressonância de Spin Eletrônica , Transporte de Elétrons , Flavina-Adenina Dinucleotídeo/análogos & derivados , Histamina , Concentração de Íons de Hidrogênio , Proteínas Ferro-Enxofre , Oxirredução
19.
Artigo em Inglês | MEDLINE | ID: mdl-17909288

RESUMO

Carbazole 1,9a-dioxygenase (CARDO) catalyzes the dihydroxylation of carbazole by angular position (C9a) carbon bonding to the imino nitrogen and its adjacent C1 carbon. CARDO consists of a terminal oxygenase component and two electron-transfer components: ferredoxin and ferredoxin reductase. The ferredoxin component of carbazole 1,9a-dioxygenase from Nocardioides aromaticivorans IC177 was crystallized at 293 K using the hanging-drop vapour-diffusion method with ammonium sulfate as the precipitant. The crystals, which were improved by macroseeding, diffract to 2.0 A resolution and belong to space group P4(1)2(1)2.


Assuntos
Proteínas de Bactérias/química , Dioxigenases/química , Ferredoxinas/química , Nocardiaceae/enzimologia , Proteínas de Bactérias/análise , Cristalização , Cristalografia por Raios X , Dioxigenases/análise , Ferredoxinas/análise
20.
Arch Microbiol ; 187(3): 217-26, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17308936

RESUMO

The extended lag period associated with vinyl chloride (VC) starvation in VC- and ethene-assimilating Nocardioides sp. strain JS614 was examined. The extended lag periods were variable (3-7 days), only associated with growth on VC or ethene, and were observed in VC- or ethene-grown cultures following 24 h carbon starvation and mid-exponential phase cultures grown on non-alkene carbon sources (e.g. acetate). Alkene monooxygenase (AkMO) and epoxyalkane:coenzyme M transferase (EaCoMT) are the initial enzymes of VC and ethene biodegradation in strain JS614. Reverse-transcription PCR confirmed that the AkMO gene etnC was expressed in response to epoxyethane, a metabolic intermediate of ethene biodegradation. Epoxyethane (0.5 mM) eliminated the extended lag period in both starved and mid-exponential phase cultures, suggesting that epoxyethane accumulation activates AkMO expression in strain JS614. AkMO activity in ethene-grown cultures was not detected after 6.7 h of carbon starvation, while 40% of the initial EaCoMT activity remained after 24 h. Acetate eliminated the extended lag period in starved cultures but not in mid-exponential phase cultures suggesting that acetate reactivates extant AkMO in starved VC- or ethene-grown cultures. The imbalance between AkMO and EaCoMT activities during starvation likely contributes to the extended lag period by delaying epoxide accumulation and subsequent AkMO induction.


Assuntos
Etilenos/metabolismo , Nocardiaceae/crescimento & desenvolvimento , Nocardiaceae/metabolismo , Cloreto de Vinil/metabolismo , Adaptação Fisiológica , Biodegradação Ambiental , Nocardiaceae/genética , Estresse Oxidativo/fisiologia
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