scholarly journals Isethionate formation from taurine in Chromohalobacter salexigens: purification of sulfoacetaldehyde reductase

Microbiology ◽  
2010 ◽  
Vol 156 (5) ◽  
pp. 1547-1555 ◽  
Author(s):  
Zdeněk Krejčík ◽  
Klaus Hollemeyer ◽  
Theo H. M. Smits ◽  
Alasdair M. Cook
Keyword(s):  
Sequence Data ◽  
Gene Clusters ◽  
Peptide Mass Fingerprinting ◽  
Rt Pcr ◽  
Regulatory Enzymes ◽  
Peptide Mass ◽  
Sds Page ◽  
Short Chain Alcohol ◽  
Chromohalobacter Salexigens ◽  
Similar Gene

Bacterial generation of isethionate (2-hydroxyethanesulfonate) from taurine (2-aminoethanesulfonate) by anaerobic gut bacteria was established in 1980. That phenomenon in pure culture was recognized as a pathway of assimilation of taurine-nitrogen. Based on the latter work, we predicted from genome-sequence data that the marine gammaproteobacterium Chromohalobacter salexigens DSM 3043 would exhibit this trait. Quantitative conversion of taurine to isethionate, identified by mass spectrometry, was confirmed, and the taurine-nitrogen was recovered as cell material. An eight-gene cluster was predicted to encode the inducible vectorial, scalar and regulatory enzymes involved, some of which were known from other taurine pathways. The genes (Csal_0153–Csal_0156) encoding a putative ATP-binding-cassette (ABC) transporter for taurine (TauAB1B2C) were shown to be inducibly transcribed by reverse transcription (RT-) PCR. An inducible taurine : 2-oxoglutarate aminotransferase [EC 2.6.1.55] was found (Csal_0158); the reaction yielded glutamate and sulfoacetaldehyde. The sulfoacetaldehyde was reduced to isethionate by NADPH-dependent sulfoacetaldehyde reductase (IsfD), a member of the short-chain alcohol dehydrogenase superfamily. The 27 kDa protein (SDS-PAGE) was identified by peptide-mass fingerprinting as the gene product of Csal_0161. The putative exporter of isethionate (IsfE) is encoded by Csal_0160; isfE was inducibly transcribed (RT-PCR). The presumed transcriptional regulator, TauR (Csal_0157), may autoregulate its own expression, typical of GntR-type regulators. Similar gene clusters were found in several marine and terrestrial gammaproteobacteria, which, in the gut canal, could be the source of not only mammalian, but also arachnid and cephalopod isethionate.

scholarly journals Purificação e avaliação da interferência de BthMP (uma metaloprotease da peçonha de Bothrops moojeni) na coagulação sanguínea

2016 ◽  
Vol 1 (2) ◽  
pp. 7
Author(s):  
Manuela Andrade Santos ◽  
Luzia Aparecida Pando ◽  
Veridiana De Melo Rodrigues ◽  
Mariana De Souza Castro ◽  
Mário Sérgio Rocha Rocha Gomes
Keyword(s):  
Peptide Mass Fingerprinting ◽  
Maldi Tof ◽  
Peptide Mass ◽  
Sds Page ◽  
Bothrops Moojeni

Neste trabalho relatamos a purificação da metaloprotease BthMP, proveniente da peçonha da serpente Bothrops moojeni. Para a purificação desta protease, utilizaram-se os passos cromatográficos de troca iônica (DEAE-Sepharose) e de exclusão molecular (Sephadex G-75), sendo o produto desses processos uma banda proteica com elevado grau de pureza, visualizada em SDS-PAGE a 14%, denominada BthMP. Esta, por sua vez, quando analisada em MALDI-TOF revelou a massa molecular nativa de 23.050 Da e 23.872 Da na forma reduzida, e a partir dos fragmentos peptídicos obtidos por Peptide Mass Fingerprinting (PMF) em MS (MALDI-TOF/TOF) indicou alta similaridade com a metaloprotease BmooMPα-I. Em termos enzimáticos, BthMP mostrou atividade proteolítica sobre azocaseína e frente ao PMSF e benzamidina, enquanto que esta atividade foi inibida na presença de EDTA, 1,10-fenantrolina e β-mercaptoetanol, sendo portanto uma metaloprotease zinco dependente da classe P-I. Ainda com este propósito, verificou-se sua especificidade enzimática sobre as cadeias Aα e Bβ do fibrinogênio, e também o consumo de fibrinogênio in vivo. Foi constatado ainda sua ação em componentes da cascata de coagulação, devido ao prolongamento do Tempo de Protrombina (TP) e do Tempo de Tromboplastina Parcial ativada (TTPa). Desta forma, a acentuada atividade fibrinogenolítica e o alto consumo de fibrinogênio in vivo são resultados que indicam a ação anticoagulante da BthMP; além do mais, sua capacidade de interferir na cascata de coagulação sugere que esta protease é promissora para futuros estudos que possam indicar um novo modelo de fármaco antitrombótico. https://doi.galoa.com.br/doi/10.17648/jibi-2448-0002-1-2-5128

Purificação e avaliação da interferência de BthMP (uma metaloprotease da peçonha de Bothrops moojeni) na coagulação sanguínea

2016 ◽  
Vol 1 (2) ◽  
Author(s):  
Manuela Andrade Santos ◽  
Luzia Aparecida Pando ◽  
Veridiana de Melo Rodrigues ◽  
Mariana de Souza Castro ◽  
Mário Sérgio Rocha Gomes
Keyword(s):  
Peptide Mass Fingerprinting ◽  
Maldi Tof ◽  
Peptide Mass ◽  
Sds Page ◽  
Bothrops Moojeni

Neste trabalho relatamos a purificação da metaloprotease BthMP, proveniente da peçonha da serpente Bothrops moojeni. Para a purificação desta protease, utilizaram-se os passos cromatográficos de troca iônica (DEAE-Sepharose) e de exclusão molecular (Sephadex G-75), sendo o produto desses processos uma banda proteica com elevado grau de pureza, visualizada em SDS-PAGE a 14%, denominada BthMP. Esta, por sua vez, quando analisada em MALDI-TOF revelou a massa molecular nativa de 23.050 Da e 23.872 Da na forma reduzida, e a partir dos fragmentos peptídicos obtidos por Peptide Mass Fingerprinting (PMF) em MS (MALDI-TOF/TOF) indicou alta similaridade com a metaloprotease BmooMPα-I. Em termos enzimáticos, BthMP mostrou atividade proteolítica sobre azocaseína e frente ao PMSF e benzamidina, enquanto que esta atividade foi inibida na presença de EDTA, 1,10-fenantrolina e β-mercaptoetanol, sendo portanto uma metaloprotease zinco dependente da classe P-I. Ainda com este propósito, verificou-se sua especificidade enzimática sobre as cadeias Aα e Bβ do fibrinogênio, e também o consumo de fibrinogênio in vivo. Foi constatado ainda sua ação em componentes da cascata de coagulação, devido ao prolongamento do Tempo de Protrombina (TP) e do Tempo de Tromboplastina Parcial ativada (TTPa). Desta forma, a acentuada atividade fibrinogenolítica e o alto consumo de fibrinogênio in vivo são resultados que indicam a ação anticoagulante da BthMP; além do mais, sua capacidade de interferir na cascata de coagulação sugere que esta protease é promissora para futuros estudos que possam indicar um novo modelo de fármaco antitrombótico.

scholarly journals Racemase activity effected by two dehydrogenases in sulfolactate degradation by Chromohalobacter salexigens: purification of (S)-sulfolactate dehydrogenase

Microbiology ◽  
2010 ◽  
Vol 156 (3) ◽  
pp. 967-974 ◽  
Author(s):  
Karin Denger ◽  
Alasdair M. Cook
Keyword(s):  
Transcriptional Regulator ◽  
Peptide Mass Fingerprinting ◽  
Uptake System ◽  
Periplasmic Binding Protein ◽  
Anion Exchange Column ◽  
Degradative Pathway ◽  
Peptide Mass ◽  
Transporter Family ◽  
Unknown Protein ◽  
Chromohalobacter Salexigens

Chromohalobacter salexigens DSM 3043, whose genome has been sequenced, is known to degrade (R,S)-sulfolactate as a sole carbon and energy source for growth. Utilization of the compound(s) was shown to be quantitative, and an eight-gene cluster (Csal_1764–Csal_1771) was hypothesized to encode the enzymes in the degradative pathway. It comprised a transcriptional regulator (SuyR), a Tripartite Tricarboxylate Transporter-family uptake system for sulfolactate (SlcHFG), two sulfolactate dehydrogenases of opposite sulfonate stereochemistry, namely novel SlcC and ComC [(R)-sulfolactate dehydrogenase] [EC1.1.1.272] and desulfonative sulfolactate sulfo-lyase (SuyAB) [EC4.4.1.24]. Inducible reduction of 3-sulfopyruvate, inducible SuyAB activity and induction of an unknown protein were detected. Separation of the soluble proteins from induced cells on an anion-exchange column yielded four relevant fractions. Two different fractions reduced sulfopyruvate with NAD(P)H, a third yielded SuyAB activity, and the fourth contained the unknown protein. The latter was identified by peptide-mass fingerprinting as SlcH, the candidate periplasmic binding protein of the transport system. Separated SuyB was also identified by peptide-mass fingerprinting. ComC was partially purified and identified by peptide-mass fingerprinting. The (R)-sulfolactate that ComC produced from sulfopyruvate was a substrate for SuyAB, which showed that SuyAB is (R)-sulfolactate sulfo-lyase. SlcC was purified to homogeneity. This enzyme also formed sulfolactate from sulfopyruvate, but the latter enantiomer was not a substrate for SuyAB. SlcC was obviously (S)-sulfolactate dehydrogenase.

Serum Protein Profiling Distinguishes BCR-ABL+ CML from Normal and Neutrophilia Cases.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4871-4871
Author(s):  
Azim M. Mohamedali ◽  
Satyaji Sahu ◽  
Nicholas Shaun B. Thomas ◽  
Ghulam J. Mufti
Keyword(s):  
Serum Proteins ◽  
Peptide Mass Fingerprinting ◽  
Protein Profiling ◽  
Sample Collection ◽  
Serum Samples ◽  
Metal Affinity ◽  
Peptide Mass ◽  
Sds Page ◽  
And Control ◽  
Normal Controls

Abstract We sought to identify additional biomarkers for chronic myelogenous leukaemia (CML) that could be an aid to early diagnosis and also yield novel antigens for immunotherapy. To this end, we screened patient serum samples at presentation against hematologically normal controls as well as patients with neutrophilia using Surface Enhanced Laser Desorption/Ionization technology (SELDI; Ciphergen ProteinChip series 4000). A total of 84 retrospective and prospective serum samples were analysed: presentation −28, reactive neutrophilia (>15x109 neutrophils/L) − 24 and hematopoietic normal controls − 33. Patients were initially screened by routine cytogenetics and in some cases with qPCR for the BCR-ABL breakpoint. The sera samples were evaluated on 4 different array surfaces and the Immobilised Metal Affinity (IMAC) array was chosen as it bound serum proteins that distinguished CML from normal controls. As little as 1 μl serum was sufficient for each analysis. Biomarker artefacts due to variations in sample collection procedures were ruled out by analysing sera (n=4) from each group at the time of collection and 3 and 6 hours post collection. There were no significant differences in any of the biomarkers at any of the time points. The spectrum of proteins obtained from each of the 84 serum samples was averaged from duplicate runs per experiment. Using the Ciphergen Express program, a panel of 5 proteins were significantly differentially expressed in CML versus the reactive neutrophilia and normal hematopoietic controls (p<0.001). These proteins were identified by a combination of purification techniques using Q HyperD F columns, desalting using reverse phase C-18 beads and isolating the biomarker by 1D-SDS PAGE. The biomarkers were identified by peptide mass fingerprinting and confirmed by Tandem MS sequencing. These were Albumin fragment − 2.8Kd (p< 3.5 x 10−5, ROC=0.78), Fibrinogen fragments − 5.3Kd (p< 6.25 x 10−10, ROC=0.07) and 5.9Kd (p< 9.6 x 10−8, ROC=0.14), Complement 3a precursor fragment − 8.9Kd (p< 0.0015, ROC= 0.70), Platelet basic protein precursor − 10.2Kd (p< 1.5 x 10−4, ROC=0.73) and Lysozyme − 14.6Kd (p=0, ROC=0.92). Biomarkers 3, 4 and 5 were also verified by antibody capture experiments using NP-20 arrays. In a blinded test set of sera, CML, normal and neutrophilia samples were correctly classified 27/28 (96%), 32/32 (100%), 20/24 (83%) respectively using a combination of the 5.3Kd, 10.2 Kd and the 14.6 Kd markers (Biomaker Pattern software). The algorithm correctly classified 21 new samples as CML (7/8) and control (10/13). The 1/8 CML was misclassified for technical reasons. Therefore, a small number of serum biomarkers in as little as 1 μl serum can be used to distinguish between patients with CML and neutrophilia or hematopoietic normal controls. Similar analyses may be applicable to other more heterogeneous hematological malignancies.

scholarly journals Molecular genetics and biochemistry of N-acetyltaurine degradation by Cupriavidus necator H16

Microbiology ◽  
2011 ◽  
Vol 157 (10) ◽  
pp. 2983-2991 ◽  
Author(s):  
Karin Denger ◽  
Sabine Lehmann ◽  
Alasdair M. Cook
Keyword(s):  
Gene Clusters ◽  
Peptide Mass Fingerprinting ◽  
Sole Source ◽  
Cupriavidus Necator ◽  
Degradative Pathway ◽  
Reverse Transcription Pcr ◽  
Peptide Mass ◽  
Coa Ligase ◽  
Cupriavidus Necator H16 ◽  
Sulfoacetaldehyde Acetyltransferase

Cupriavidus necator H16 (DSM 428), whose genome has been sequenced, was found to degrade N-acetyltaurine as a sole source of carbon and energy for growth. Utilization of the compound was quantitative. The degradative pathway involved an inducible N-acetyltaurine amidohydrolase (NaaS), which catalysed the cleavage of N-acetyltaurine to acetate and taurine. The degradation of the latter compound is via an inducible, degradative pathway that involves taurine dehydrogenase [EC 1.4.2.–], sulfoacetaldehyde acetyltransferase [EC 2.3.3.15], phosphotransacetylase [EC 2.4.1.8], a sulfite exporter [TC 9.A.29.2.1] and sulfite dehydrogenase [EC 1.8.2.1]. Induction of the expression of representative gene products, encoded by at least four gene clusters, was confirmed biochemically. The acetate released by NaaS was activated to acetyl-CoA by an inducible acetate–CoA ligase [EC 6.2.1.1]. NaaS was purified to homogeneity; it had a K m value of 9.4 mM for N-acetyltaurine, and it contained tightly bound Zn and Fe atoms. The denatured enzyme has a molecular mass of about 61 kDa (determined by SDS-PAGE) and the native enzyme was apparently monomeric. Peptide-mass fingerprinting identified the locus tag as H16_B0868 in a five-gene cluster, naaROPST (H16_B0865–H16_B0869). The cluster presumably encodes a LysR-type transcriptional regulator (NaaR), a membrane protein (NaaO), a solute : sodium symporter-family permease [TC 2.A.21] (NaaP), the metal-dependent amidohydrolase (NaaS) and a putative metallochaperone (COG0523) (NaaT). Reverse-transcription PCR indicated that naaOPST were inducibly transcribed.

Introductory Glycosylation Analysis Using SDS-PAGE and Peptide Mass Fingerprinting

Glycomics ◽  
2008 ◽  
pp. 205-212 ◽  
Author(s):  
Nicole Wilson ◽  
Raina Simpson ◽  
Catherine Cooper-Liddell
Keyword(s):  
Peptide Mass Fingerprinting ◽  
Peptide Mass ◽  
Sds Page

Proteomic Analysis of Medicinal Plant Calotropis Gigantea by insilico Peptide Mass Fingerprinting

2020 ◽  
Vol 16 ◽  
Author(s):  
Saad Ur Rehman ◽  
Muhammad Rizwan ◽  
Sajid Khan ◽  
Azhar Mehmood ◽  
Anum Munir
Keyword(s):  
Medicinal Plant ◽  
Structure Prediction ◽  
3D Structure ◽  
Peptide Mass Fingerprinting ◽  
Protein Docking ◽  
Receptor Protein ◽  
Human Leukemia ◽  
Protein Database ◽  
Calotropis Gigantea ◽  
Peptide Mass

: Medicinal plants are the basic source of medicinal compounds traditionally used for the treatment of human diseases. Calotropis gigantea a medicinal plant belonging to the family of Apocynaceae in the plant kingdom and subfamily Asclepiadaceae usually bearing multiple medicinal properties to cure a variety of diseases. Background: The Peptide Mass Fingerprinting (PMF) identifies the proteins from a reference protein database by comparing the amino acid sequence that is previously stored in a database and identified. Method: The calculation of insilico peptide masses is done through the ExPASy PeptideMass and these masses are used to identify the peptides from MASCOT online server. Anticancer probability is calculated from the iACP server, docking of active peptides is done by CABS-dock the server. Objective: The purpose of the study is to identify the peptides having anti-cancerous properties by in-silico peptide mass fingerprinting. Results : The anti-cancerous peptides are identified with the MASCOT peptide mass fingerprinting server, the identified peptides are screened and only the anti-cancer are selected. De novo peptide structure prediction is used for 3D structure prediction by PEP-FOLD 3 server. The docking results confirm strong bonding with the interacting amino acids of the receptor protein of breast cancer BRCA1 which shows the best peptide binding to the Active chain, the human leukemia protein docking with peptides shows the accurate binding. Conclusion : These peptides are stable and functional and are the best way for the treatment of cancer and many other deadly diseases.

scholarly journals Identification of Putative Biosynthetic Gene Clusters for Tolyporphins in Multiple Filamentous Cyanobacteria

Life ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 758
Author(s):  
Xiaohe Jin ◽  
Yunlong Zhang ◽  
Ran Zhang ◽  
Kathy-Uyen Nguyen ◽  
Jonathan S. Lindsey ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Filamentous Cyanobacterium ◽  
Biosynthetic Gene ◽  
Filamentous Cyanobacteria ◽  
Biosynthetic Gene Clusters ◽  
Common Component ◽  
Homology Searching ◽  
Similar Gene

Tolyporphins A–R are unusual tetrapyrrole macrocycles produced by the non-axenic filamentous cyanobacterium HT-58-2. A putative biosynthetic gene cluster for biosynthesis of tolyporphins (here termed BGC-1) was previously identified in the genome of HT-58-2. Here, homology searching of BGC-1 in HT-58-2 led to identification of similar BGCs in seven other filamentous cyanobacteria, including strains Nostoc sp. 106C, Nostoc sp. RF31YmG, Nostoc sp. FACHB-892, Brasilonema octagenarum UFV-OR1, Brasilonema octagenarum UFV-E1, Brasilonema sennae CENA114 and Oculatella sp. LEGE 06141, suggesting their potential for tolyporphins production. A similar gene cluster (BGC-2) also was identified unexpectedly in HT-58-2. Tolyporphins BGCs were not identified in unicellular cyanobacteria. Phylogenetic analysis based on 16S rRNA and a common component of the BGCs, TolD, points to a close evolutionary history between each strain and their respective tolyporphins BGC. Though identified with putative tolyporphins BGCs, examination of pigments extracted from three cyanobacteria has not revealed the presence of tolyporphins. Overall, the identification of BGCs and potential producers of tolyporphins presents a collection of candidate cyanobacteria for genetic and biochemical analysis pertaining to these unusual tetrapyrrole macrocycles.

scholarly journals The Fish Pathogen Vibrio ordalii Under Iron Deprivation Produces the Siderophore Piscibactin

2019 ◽  
Vol 7 (9) ◽  
pp. 313 ◽  
Author(s):  
Pamela Ruiz ◽  
Miguel Balado ◽  
Juan Carlos Fuentes-Monteverde ◽  
Alicia E. Toranzo ◽  
Jaime Rodríguez ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Iron Uptake ◽  
Peptide Mass Fingerprinting ◽  
Iron Limitation ◽  
Fish Pathogen ◽  
Fish Pathogens ◽  
Iron Deprivation ◽  
Peptide Mass ◽  
Photobacterium Damselae ◽  
Vibrio Ordalii

Vibrio ordalii is the causative agent of vibriosis, mainly in salmonid fishes, and its virulence mechanisms are still not completely understood. In previous works we demonstrated that V. ordalii possess several iron uptake mechanisms based on heme utilization and siderophore production. The aim of the present work was to confirm the production and utilization of piscibactin as a siderophore by V. ordalii. Using genetic analysis, identification by peptide mass fingerprinting (PMF) of iron-regulated membrane proteins and chemical identification by LC-HRMS, we were able to clearly demonstrate that V. ordalii produces piscibactin under iron limitation. The synthesis and transport of this siderophore is encoded by a chromosomal gene cluster homologous to another one described in V. anguillarum, which also encodes the synthesis of piscibactin. Using β-galactosidase assays we were able to show that two potential promoters regulated by iron control the transcription of this gene cluster in V. ordalii. Moreover, biosynthetic and transport proteins corresponding to piscibactin synthesis and uptake could be identified in membrane fractions of V. ordalii cells grown under iron limitation. The synthesis of piscibactin was previously reported in other fish pathogens like Photobacterium damselae subsp. piscicida and V. anguillarum, which highlights the importance of this siderophore as a key virulence factor in Vibrionaceae bacteria infecting poikilothermic animals.

scholarly journals Optimization of Protein Extraction Method for 2DE Proteomics of Goat’s Milk

Molecules ◽  
2020 ◽  
Vol 25 (11) ◽  
pp. 2625
Author(s):  
Muzammeer Mansor ◽  
Jameel R. Al-Obaidi ◽  
Nurain Nadiah Jaafar ◽  
Intan Hakimah Ismail ◽  
Atiqah Farah Zakaria ◽  
...  
Keyword(s):  
Extraction Method ◽  
Protein Extraction ◽  
Chloroform Solution ◽  
Milk Proteins ◽  
Peptide Mass Fingerprinting ◽  
Extraction Methods ◽  
Dairy Goats ◽  
Goat’S Milk ◽  
Peptide Mass ◽  
Goat's Milk

Two-dimensional electrophoretic (2DE)-based proteomics remains a powerful tool for allergenomic analysis of goat’s milk but requires effective extraction of proteins to accurately profile the overall causative allergens. However, there are several current issues with goat’s milk allergenomic analysis, and among these are the absence of established standardized extraction method for goat’s milk proteomes and the complexity of goat’s milk matrix that may hamper the efficacy of protein extraction. This study aimed to evaluate the efficacies of three different protein extraction methods, qualitatively and quantitatively, for the 2DE-proteomics, using milk from two commercial dairy goats in Malaysia, Saanen, and Jamnapari. Goat’s milk samples from both breeds were extracted by using three different methods: a milk dilution in urea/thiourea based buffer (Method A), a triphasic separation protocol in methanol/chloroform solution (Method B), and a dilution in sulfite-based buffer (Method C). The efficacies of the extraction methods were assessed further by performing the protein concentration assay and 1D and 2D SDS-PAGE profiling, as well as identifying proteins by MALDI-TOF/TOF MS/MS. The results showed that method A recovered the highest amount of proteins (72.68% for Saanen and 71.25% for Jamnapari) and produced the highest number of protein spots (199 ± 16.1 and 267 ± 10.6 total spots for Saanen and Jamnapari, respectively) with superior gel resolution and minimal streaking. Six milk protein spots from both breeds were identified based on the positive peptide mass fingerprinting matches with ruminant milk proteins from public databases, using the Mascot software. These results attest to the fitness of the optimized protein extraction protocol, method A, for 2DE proteomic and future allergenomic analysis of the goat’s milk.

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