Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 5 de 5
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Int J Biol Macromol ; 171: 465-479, 2021 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-33428952

RESUMO

The ubiquitous nature of hemoglobins, their presence in multiple forms and low cellular expression in organisms suggests alternative physiological functions of hemoglobins in addition to oxygen transport and storage. Previous research has proposed enzymatic function of hemoglobins such as nitric oxide dioxygenase, nitrite reductase and hydroxylamine reductase. In all these enzymatic functions, active ferrous form of hemoglobin is converted to ferric form and reconversion of ferric to ferrous through reduction partners is under active investigation. The model alga C. reinhardtii contains multiple globins and is thus expected to have multiple putative methemoglobin reductases to augment the physiological functions of the novel hemoglobins. In this regard, three putative methemoglobin reductases and three algal hemoglobins were characterized. Our results signify that the identified putative methemoglobin reductases can reduce algal methemoglobins in a nonspecific manner under in vitro conditions. Enzyme kinetics of two putative methemoglobin reductases with methemoglobins as substrates and in silico analysis support interaction between the hemoglobins and the two reduction partners as also observed in vitro. Our investigation on algal methemoglobin reductases underpins the valuable chemistry of nitric oxide with the newly discovered hemoglobins to ensure their physiological relevance, with multiple hemoglobins probably necessitating the presence of multiple reductases.


Assuntos
Chlamydomonas reinhardtii/enzimologia , Citocromo-B(5) Redutase/fisiologia , Oxigenases/metabolismo , Proteínas de Plantas/fisiologia , Hemoglobinas Truncadas/metabolismo , Técnicas de Química Analítica , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Sequência Conservada , Citocromo-B(5) Redutase/química , Citocromo-B(5) Redutase/genética , Citocromo-B(5) Redutase/isolamento & purificação , Humanos , Cinética , Metemoglobina/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Oxirredução , Proteínas de Plantas/isolamento & purificação , Conformação Proteica , Domínios Proteicos , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Eletricidade Estática , Especificidade por Substrato , Hemoglobinas Truncadas/genética , Hemoglobinas Truncadas/isolamento & purificação
2.
Biochim Biophys Acta ; 1840(7): 2281-8, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24704259

RESUMO

BACKGROUND: Understanding the molecular mechanism through which proteins are functional at extreme high and low temperatures is one of the key issues in structural biology. To investigate this phenomenon, we have focused on two instructive truncated hemoglobins from Thermobifida fusca (Tf-trHbO) and Mycobacterium tuberculosis (Mt-trHbO); although the two proteins are structurally nearly identical, only the former is stable at high temperatures. METHODS: We used molecular dynamics simulations at different temperatures as well as thermal melting profile measurements of both wild type proteins and two mutants designed to interchange the amino acid residue, either Pro or Gly, at E3 position. RESULTS: The results show that the presence of a Pro at the E3 position is able to increase (by 8°) or decrease (by 4°) the melting temperature of Mt-trHbO and Tf-trHbO, respectively. We observed that the ProE3 alters the structure of the CD loop, making it more flexible. CONCLUSIONS: This gain in flexibility allows the protein to concentrate its fluctuations in this single loop and avoid unfolding. The alternate conformations of the CD loop also favor the formation of more salt-bridge interactions, together augmenting the protein's thermostability. GENERAL SIGNIFICANCE: These results indicate a clear structural and dynamical role of a key residue for thermal stability in truncated hemoglobins.


Assuntos
Modelos Moleculares , Mycobacterium tuberculosis/metabolismo , Estabilidade Proteica , Hemoglobinas Truncadas/química , Actinomycetales/química , Actinomycetales/metabolismo , Temperatura Alta , Humanos , Simulação de Dinâmica Molecular , Mycobacterium tuberculosis/química , Hemoglobinas Truncadas/isolamento & purificação , Hemoglobinas Truncadas/metabolismo
3.
Protein Expr Purif ; 86(1): 21-6, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22963793

RESUMO

Vitreoscilla hemoglobin (VHb) was widely used in metabolic engineering to improve oxygen utilization in the low oxygen environment. It is sometimes necessary to remove affinity tags because they may impede functions of target proteins. Here we report an efficient method employing Glutamate-specific endopeptidase from Bacillus licheformis (GSE-BL) to perform the cleavage between VHb and His-tag. The optimal length of GSE-BL treatment was 15min. Results of SDS-PAGE and western blot demonstrated that the His-tag of VHb-His(6) was nearly completely removed, the purity of VHb was enhanced from 74% to 99.5%, and the yield of tagless VHb from VHb-His(6) was 92.2%. Results of CO difference spectrum suggested that tagless VHb was more prone to bind to CO compared with VHb-His(6). It was observed that tagless VHb displayed higher catalase activity than VHb-His(6). The enhancement of welan gum yield was more significant by addition of tagless VHb compared with addition of VHb-His(6). This method can be utilized to mass-produce tagless VHb, thus widening the application of VHb in various industries.


Assuntos
Bacillus/enzimologia , Proteínas de Bactérias/isolamento & purificação , Endopeptidases/química , Ácido Glutâmico/química , Hemoglobinas Truncadas/isolamento & purificação , Vitreoscilla/química , Marcadores de Afinidade , Bacillus/química , Proteínas de Bactérias/química , Western Blotting , Catalase/química , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Ensaios Enzimáticos , Plasmídeos/química , Polissacarídeos Bacterianos/química , Ligação Proteica , Proteólise , Proteínas Recombinantes/química , Fatores de Tempo , Hemoglobinas Truncadas/química
4.
Methods Enzymol ; 436: 255-72, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18237637

RESUMO

The use of the heterologous bacterial hemoglobin (VHb) from Vitreoscilla to enhance growth and productivity of Escherichia coli under conditions of oxygen limitation has been one of the foremost examples of metabolic engineering. Although VHb has earned its merits during the last two decades by providing enhanced physiological enhancements to organisms from all kingdoms of life, it has been the candidate of choice primarily for historical reasons. Findings made during the last years, however, suggest that hemoglobin and flavohemoglobin proteins from bacterial species other than Vitreoscilla or artificially generated mutant proteins or fusion variants of hemoglobins and flavohemoglobins may be better suited for use in biotechnological processes. This account provides guidelines for the assessment of biotechnologically relevant characteristics conferred by such novel heterologous hemoglobins and flavohemoglobins in E. coli.


Assuntos
Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/isolamento & purificação , Hemoglobinas Truncadas/biossíntese , Hemoglobinas Truncadas/isolamento & purificação , Proteínas de Bactérias/genética , Reatores Biológicos , Biotecnologia , Western Blotting , Meios de Cultura , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Expressão Gênica , Genes Bacterianos , Heme/metabolismo , Oxigênio/metabolismo , Plasmídeos/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Espectrofotometria , Hemoglobinas Truncadas/genética , Vitreoscilla/química , Vitreoscilla/genética
5.
Methods Enzymol ; 436: 289-302, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18237639

RESUMO

Campylobacter jejuni is a Gram-negative microaerophilic bacterium that occurs as a common gut commensal in many food-producing animals and birds. Contamination of meat during processing is an important route of transmission, and C. jejuni is now recognized as one of the most important causes of bacterial gastroenteritis worldwide. C. jejuni is notable, but not unique, in possessing two different hemoglobins. The first is termed Cgb and is a single-domain hemoglobin (i.e., having no other protein domain or cofactor) with clear structural similarities (3/3) with myoglobin, the heme domain of flavohemoglobins and Vitreoscilla hemoglobin. It is well established that Cgb plays a key role in providing resistance to C. jejuni in the face of NO and other reactive nitrogen species that might be encountered in its environments. The second globin is Ctb, a truncated globin (2/2trHb) in class III, until recently the least well-understood class of these ubiquitous globins. In C. jejuni, both globin genes are members of a small regulon activated by the NssR protein, which acts as an NO sensor and transcriptional regulator. In this contribution, we describe the cloning of both the cgb and ctb genes from C. jejuni chromosomal DNA, construction of expression vectors in E. coli, and a simple purification procedure for each globin. A brief account of the spectroscopic characteristics of both globins is presented.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Campylobacter jejuni/química , Campylobacter jejuni/genética , Hemoglobinas Truncadas/genética , Hemoglobinas Truncadas/isolamento & purificação , Sequência de Bases , Campylobacter jejuni/patogenicidade , Clonagem Molecular , Primers do DNA/genética , Escherichia coli/genética , Microbiologia de Alimentos , Expressão Gênica , Genes Bacterianos , Óxido Nítrico/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Espectrofotometria
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...