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1.
Appl Microbiol Biotechnol ; 99(13): 5475-85, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25575887

RESUMO

Improving the health beneficial fatty acid content of meat and milk is a major challenge requiring an increased understanding of rumen lipid metabolism. In this study, we isolated and characterized rumen bacterial lipases/esterases using functional metagenomics. Metagenomic libraries were constructed from DNA extracted from strained rumen fluid (SRF), solid-attached bacteria (SAB) and liquid-associated rumen bacteria (LAB), ligated into a fosmid vector and subsequently transformed into an Escherichia coli host. Fosmid libraries consisted of 7,744; 8,448; and 7,680 clones with an average insert size of 30 to 35 kbp for SRF, SAB and LAB, respectively. Transformants were screened on spirit blue agar plates containing tributyrin for lipase/esterase activity. Five SAB and four LAB clones exhibited lipolytic activity, and no positive clones were found in the SRF library. Fosmids from positive clones were pyrosequenced and twelve putative lipase/esterase genes and two phospholipase genes retrieved. Although the derived proteins clustered into diverse esterase and lipase families, a degree of novelty was seen, with homology ranging from 40 to 78% following BlastP searches. Isolated lipases/esterases exhibited activity against mostly short- to medium-chain substrates across a range of temperatures and pH. The function of these novel enzymes recovered in ruminal metabolism needs further investigation, alongside their potential industrial uses.


Assuntos
Esterases/genética , Esterases/metabolismo , Metagenoma , Rúmen/microbiologia , Animais , Bovinos , Escherichia coli/genética , Esterases/isolamento & purificação , Expressão Gênica , Concentração de Íons de Hidrogênio , Hidrólise , Dados de Sequência Molecular , Análise de Sequência de DNA , Temperatura , Transformação Bacteriana , Triglicerídeos/metabolismo
2.
Arch Biochem Biophys ; 484(1): 8-15, 2009 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-19467626

RESUMO

Pseudomonas lupanine hydroxylase is a periplasmic-localised, two domain quinocytochrome c enzyme. It requires numerous post-translocation modifications involving signal peptide processing, disulphide bridge formation and, heme linkage in the carboxy-terminal cytochrome c domain to eventually generate a Ca(2+)-bound quino-c hemoprotein that hydroxylates the plant alkaloid, lupanine. An exported, functional recombinant enzyme was generated in Escherichia coli by co-expression with cytochrome c maturation factors. Increased growth temperatures ranging from 18 to 30 degrees C gradually raised the enzyme production to a peak together with its concomitant aggregation as red solid particles, readily activatable in a fully functional form by mild chaotropic treatment. Here, we demonstrate that the exported lupanine hydroxylase undergoes a cascade transition from a soluble to "non-classical" inclusion body form when build-up in the periplasm exceeded a basal threshold concentration. These periplasmic aggregates were distinct from the non-secreted, signal-sequenceless counterpart that occurred as misfolded, non-functional concatamers in the form of classical inclusion bodies. We discuss our findings in the light of current models of how aggregation of lupanine hydroxylase arises in the periplasmic space.


Assuntos
Escherichia coli/enzimologia , Corpos de Inclusão/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Periplasma/enzimologia , Sequência de Bases , Primers do DNA , Dissulfetos/metabolismo , Ativação Enzimática , Microscopia Eletrônica , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/isolamento & purificação , Transporte Proteico , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Solubilidade
3.
Mol Membr Biol ; 25(5): 388-99, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18651317

RESUMO

Proteins destined for translocation across the prokaryotic cytoplasmic membrane are synthesized as precursors carrying transient N-terminal extensions known as signal sequences. They facilitate initial engagement of precursor proteins with the sec-dependent translocase to initiate active threading of the polypeptide across the membrane. The translocated precursor is then processed by a transcytoplasmic signal peptidase anchored to the inner membrane. The temporal nature of cleavage of the signal sequence during pre-protein translocation has remained elusive. Using an engineered mammalian cytochrome b(5) precursor we demonstrate that the signal peptide processing in Escherichia coli is an event that can occur after almost complete exocytoplasmic translocation of the preprotein is accomplished. We discuss implications of the findings in light of the known working model of sec-dependent pre-protein translocon.


Assuntos
Citocromos b5/metabolismo , Precursores Enzimáticos/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Proteínas de Membrana/metabolismo , Sinais Direcionadores de Proteínas/fisiologia , Serina Endopeptidases/metabolismo , Animais , Membrana Celular/genética , Membrana Celular/metabolismo , Citocromos b5/genética , Precursores Enzimáticos/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Mamíferos , Proteínas de Membrana/genética , Engenharia de Proteínas/métodos , Transporte Proteico/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/genética
4.
Protein Sci ; 13(9): 2458-69, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15322285

RESUMO

A directed evolutionary approach is described that searches short, random peptide sequences for appendage at the secretory signal peptide-mature protein junction to seek ideal algorithms for both efficient and hyper export of recombinant proteins to the periplasm of Escherichia coli. The strategy employs simple, visual detection of positive clones using a PINK expression system that faithfully reports on export status of a mammalian hemoprotein in E. coli. With-in "sequence spaces" ranging from 1 to 13 residues, a significant but highly variable secretory fitness was scored such that the rate of secretion reciprocally correlated with the membrane-associated precursor pool of the evolved exportable hemoproteins. Three clusters of hyper, median, and hypo exporters were isolated. These had corresponding net charges of -1, 0, and +1 within the evolved sequence space, which in turn clearly correlated with the prevailing magnitude and polarity of the membrane energization states. The findings suggest that both the nature of the charged residue and the proximal sequence in the early mature region are the crucial determinants of the protonophore-dependent electrophoretic discharge of the precursor across the inner membrane of E. coli. We conclude that the directed evolutionary approach will find ready application in engineering recombinant proteins for their efficient secretion via the sec export pathway in E. coli.


Assuntos
Engenharia de Proteínas/métodos , Precursores de Proteínas/metabolismo , Sinais Direcionadores de Proteínas , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Evolução Biológica , Membrana Celular/metabolismo , Citocromos b5/genética , Citocromos b5/metabolismo , Citoplasma/metabolismo , Escherichia coli/genética , Hemeproteínas/genética , Hemeproteínas/metabolismo , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Periplasma/genética , Periplasma/metabolismo , Precursores de Proteínas/genética , Sinais Direcionadores de Proteínas/genética , Transporte Proteico , Proteínas Recombinantes/genética , Serina Endopeptidases/metabolismo
5.
PLoS One ; 8(8): e69076, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23950883

RESUMO

Following the isolation, cultivation and characterization of the rumen bacterium Anaerovibrio lipolyticus in the 1960s, it has been recognized as one of the major species involved in lipid hydrolysis in ruminant animals. However, there has been limited characterization of the lipases from the bacterium, despite the importance of understanding lipolysis and its impact on subsequent biohydrogenation of polyunsaturated fatty acids by rumen microbes. This study describes the draft genome of Anaerovibrio lipolytica 5ST, and the characterization of three lipolytic genes and their translated protein. The uncompleted draft genome was 2.83 Mbp and comprised of 2,673 coding sequences with a G+C content of 43.3%. Three putative lipase genes, alipA, alipB and alipC, encoding 492-, 438- and 248- amino acid peptides respectively, were identified using RAST. Phylogenetic analysis indicated that alipA and alipB clustered with the GDSL/SGNH family II, and alipC clustered with lipolytic enzymes from family V. Subsequent expression and purification of the enzymes showed that they were thermally unstable and had higher activities at neutral to alkaline pH. Substrate specificity assays indicated that the enzymes had higher hydrolytic activity against caprylate (C8), laurate (C12) and myristate (C14).


Assuntos
Lipase/genética , Lipase/metabolismo , Veillonellaceae/enzimologia , Veillonellaceae/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sequência Conservada , Ativação Enzimática , Expressão Gênica , Concentração de Íons de Hidrogênio , Lipase/química , Lipase/isolamento & purificação , Lipólise , Dados de Sequência Molecular , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Especificidade por Substrato , Temperatura , Veillonellaceae/classificação
6.
FEMS Microbiol Lett ; 334(1): 35-43, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22681241

RESUMO

Lupanine hydroxylase (LH), a quinohaemoprotein, catabolizes lupanine and possesses four cysteine (Cys) residues; two associated with a cytochrome c motif ((586)Cys and (589)Cys), while the role of the remaining two residues ((124)Cys and (143)Cys) is unclear. Structural graphic simulation using homology modelling suggested a potential second -S-S- bond, a common feature between adjacent Cys residues in other quinohaemoproteins; however, in LH, these residues are located 18 amino acids apart. Formation of the second disulphide bond was initially chemically confirmed by iodomethane alkylation with 91% loss of enzymic activity, and no significant change was observed with unreduced alkylated protein. Dithiothreitol-induced reduction of LH followed by Cd(2+) treatment also resulted in significant loss of activity in a dose-dependent manner. Subsequent investigation into the role of disulphide bond in LH was performed using engineered (143)Cys→Ser and (124,143)Cys→Ser mutants and exhibited 25% and zero activity, respectively, of wild type in the periplasm. Homology structure prediction showed three changes in α-helices and four in ß-pleated sheets in (143)Cys→Ser mutant, and (124,143)Cys→Ser mutant had six changes in α-helices and nine in ß-pleated sheets. These mutations resulted in the enlargement of the molecule and affect the enzyme activity because of structural changes in the cytochrome c domain.


Assuntos
Proteínas de Bactérias/genética , Escherichia coli/genética , Mutação de Sentido Incorreto , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Pseudomonas/enzimologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Escherichia coli/metabolismo , Expressão Gênica , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/química , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Estrutura Terciária de Proteína , Pseudomonas/química , Pseudomonas/genética
7.
Protein Sci ; 19(7): 1344-53, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20506367

RESUMO

A chimeric mammalian globular cytochrome b(5) fused to Escherichia coli alkaline phosphatase signal sequence (SS) was used as a model probe to investigate the influence of substituting each one of the standard 20 amino acids at its N-terminus on the Sec-dependent export of the precursor to the periplasmic space of E. coli. Substituting the native Met(+1) of the passenger protein flanking the SS with any one of the remaining 19 amino acids introduced significant changes in the export of cytochrome b(5) without jamming the Sec-dependent translocon. Acidic and hydrophilic residues proved to be the most efficient promoters of export. Small, nonbulky and basic residues yielded intermediate levels of the hemoprotein export. Replacement with a Cys(+1) residue generated significant quantities of both monomeric and disulfide-linked dimeric forms. However, bulky, aromatic and hydrophobic residues caused a significant decline in the rates of secretion. In expectation with their absences in the natural periplasmically secreted proteins, Pro and Ile-tagged cytochrome b(5) precursors failed to generate any detectable secreted recombinant products. Although Ala, amongst the native E. coli periplasmic proteins, is the preferred X(+1) residue with an occurrence of 50% frequency, it proved half as effective in promoting export when inserted proximally to the SS of cytochrome b(5). The mechanisms involved for these export variations are discussed. The findings will prove beneficial for high-level generation of recombinant proteins by secretory means for pharmaceutical and related biotechnological applications.


Assuntos
Citocromos b5/química , Citocromos b5/metabolismo , Escherichia coli/metabolismo , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Sinais Direcionadores de Proteínas/fisiologia , Animais , Citocromos b5/genética , Escherichia coli/genética , Humanos , Precursores de Proteínas/genética , Sinais Direcionadores de Proteínas/genética , Transporte Proteico/genética , Transporte Proteico/fisiologia
8.
Arch Biochem Biophys ; 412(2): 259-66, 2003 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-12667490

RESUMO

Sequence analyses of the C-terminal membrane intercalative region of the rat cytochrome b(5) indicated that this domain has, in addition to a signal sequence, a combined element of the classic stop-transfer sequence typically found in a variety of transmembrane proteins. Such bitopic protein arrangements arise by tandem but topogenically displaced activities of cleavable/noncleavable signal and stop-transfer sequences. A fusion precursor comprising an N-terminally linked prokaryotic signal sequence and the full-length of mammalian cytochrome b(5), including its C-terminal membrane insertion sequence, was engineered to investigate the outcome of this combination of signals on the targeting and topology of the cytochrome b(5) in the endoplasmic reticulum membrane. Precytochrome b(5) was cotranslationally translocated across the endoplasmic reticulum membrane. The signal-processed cytochrome b(5) was integrally anchored in the membrane with the globular domain facing the lumen. Thus, the topology of the signal sequence-directed cytochrome b(5) in the microsomal vesicle was reversed with respect to that of the native form. Posttranslational incubation of the precytochrome b(5) with microsomes resulted in a "loose" incorporation of the unprocessed form onto the surface of the vesicle. Our findings suggest that the membrane-insertion sequence of cytochrome b(5) has a functional stop-transfer sequence. We discuss the implications of these findings with respect to selective targeting of cytochrome b(5) to the endoplasmic reticulum membrane in the view that signal and stop-transfer sequences are often interchangeable or combined for topogenic functions.


Assuntos
Citocromos b5/química , Citocromos b5/metabolismo , Retículo Endoplasmático/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Citocromos b5/genética , DNA/genética , Humanos , Técnicas In Vitro , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microssomos/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Estrutura Molecular , Sinais Direcionadores de Proteínas/genética , Estrutura Terciária de Proteína , Ratos , Transdução de Sinais
9.
Comp Funct Genomics ; 4(4): 376-91, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-18629082

RESUMO

We sought to test the hypothesis that mutant bacterial strains could be discriminated from each other on the basis of the metabolites they secrete into the medium (their 'metabolic footprint'), using two methods of 'global' metabolite analysis (FT-IR and direct injection electrospray mass spectrometry). The biological system used was based on a published study of Escherichia coli tryptophan mutants that had been analysed and discriminated by Yanofsky and colleagues using transcriptome analysis. Wild-type strains supplemented with tryptophan or analogues could be discriminated from controls using FT-IR of 24 h broths, as could each of the mutant strains in both minimal and supplemented media. Direct injection electrospray mass spectrometry with unit mass resolution could also be used to discriminate the strains from each other, and had the advantage that the discrimination required the use of just two or three masses in each case. These were determined via a genetic algorithm. Both methods are rapid, reagentless, reproducible and cheap, and might beneficially be extended to the analysis of gene knockout libraries.

10.
J Biol Chem ; 278(46): 45555-62, 2003 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-12930844

RESUMO

This report suggests an important physiological role of a CYP in the accumulation of uroporphyrin I arising from catalytic oxidative conversion of uroporphyrinogen I to uroporphyrin I in the periplasm of Escherichia coli cultured in the presence of 5-aminolevulinic acid. A structurally competent Streptomyces griseus CYP105D1 was expressed as an engineered, exportable form in aerobically grown E. coli. Its progressive induction in the presence of 5-aminolevulinic acid-supplemented medium was accompanied by an accumulation of a greater than 100-fold higher amount of uroporphyrin I in the periplasm relative to cells lacking CYP105D1. Expression of a cytoplasm-resident engineered CYP105D1 at a comparative level to the secreted form was far less effective in promoting porphyrin accumulation in the periplasm. Expression at a 10-fold molar excess over the exported CYP105D1 of another periplasmically exported hemoprotein, the globular core of cytochrome b5, did not substitute the role of the periplasmically localized CYP105D1 in promoting porphyrin production. This, therefore, eliminated the possibility that uroporphyrin accumulation is merely a result of increased hemoprotein synthesis. Moreover, in the strain that secreted CYP105D1, uroporphyrin production was considerably reduced by azole-based P450 inhibitors. Production of both holo-CYP105D1 and uroporphyrin was dependent upon 5-aminolevulinic acid, except that at higher concentrations this resulted in a decrease in uroporphyrin. This study suggests that the exported CYP105D1 oxidatively catalyzes periplasmic conversion of uroporphyrinogen I to uroporphyrin I in E. coli. The findings have significant implications in the ontogenesis of human uroporphyria-related diseases.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Escherichia coli/enzimologia , Periplasma/metabolismo , Uroporfirinas/metabolismo , Ácido Aminolevulínico/metabolismo , Sistema Enzimático do Citocromo P-450/química , Citocromos b5/metabolismo , Citoplasma/metabolismo , DNA/metabolismo , Relação Dose-Resposta a Droga , Escherichia coli/metabolismo , Heme/química , Humanos , Concentração de Íons de Hidrogênio , Modelos Biológicos , Oxigênio/metabolismo , Porfirinas/metabolismo , Espectrofotometria , Streptomyces griseus/enzimologia
11.
Appl Environ Microbiol ; 69(8): 4727-31, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12902264

RESUMO

We have cloned, sequenced, and heterologously expressed a periplasmic cytochrome c from a lupanine-utilizing Pseudomonas putida strain. Aerobic batch cultivation of Escherichia coli TB1 harboring the cytochrome c gene placed downstream of the lac promoter in pUC9 vector resulted in significant production of the holo-cytochrome c in the periplasm ( approximately 4 mg of hemoprotein/liter of culture). The recombinant cytochrome c was purified to homogeneity and was found to be functional in accepting electrons from lupanine hydroxylase while catalyzing hydroxylation of lupanine. Comparison of the N-terminal amino acid sequence of the isolated cytochrome c with that deduced from the DNA sequence indicated that the signal sequence was processed at the bond position predicted by the SigPep program. The molecular size of the cytochrome c determined by electrospray mass spectrometry (9,595) was in precise agreement with that predicted from the nucleotide sequence.


Assuntos
Alcaloides/metabolismo , Proteínas de Bactérias/metabolismo , Grupo dos Citocromos c/metabolismo , Escherichia coli/genética , Periplasma/enzimologia , Pseudomonas putida/enzimologia , Aerobiose , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Grupo dos Citocromos c/química , Grupo dos Citocromos c/genética , Grupo dos Citocromos c/isolamento & purificação , Dados de Sequência Molecular , Transporte Proteico , Proteínas Recombinantes/metabolismo , Esparteína/análogos & derivados
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