scholarly journals Characterization of the 4-Hydroxybenzoyl-Coenzyme A Thioesterase from Arthrobacter sp. Strain SU

2003 ◽  
Vol 69 (5) ◽  
pp. 2707-2711 ◽  
Author(s):  
Zhihao Zhuang ◽  
Karl-Heinz Gartemann ◽  
Rudolf Eichenlaub ◽  
Debra Dunaway-Mariano
Keyword(s):  
Coenzyme A ◽  
Sequence Data ◽  
Protein A ◽  
Coa Ligase ◽  
Protein Sequence Data ◽  
Coa Thioesters ◽  
Ph Range ◽  
Rate Profile ◽  
Encoding Genes

ABSTRACT The Arthrobacter sp. strain SU 4-chlorobenzoate (4-CBA) dehalogenation pathway converts 4-CBA to 4-hydroxybenzoate (4-HBA). The pathway operon contains the genes fcbA, fcbB, and fcbC (A. Schmitz, K. H. Gartemann, J. Fiedler, E. Grund, and R. Eichenlaub, Appl. Environ. Microbiol. 58:4068-4071, 1992). Genes fcbA and fcbB encode 4-CBA-coenzyme A (CoA) ligase and 4-CBA-CoA dehalogenase, respectively, whereas the function of fcbC is not known. We subcloned fcbC and expressed it in Escherichia coli, and we purified and characterized the FcbC protein. A substrate activity screen identified benzoyl-CoA thioesters as the most active substrates. Catalysis of 4-HBA-CoA hydrolysis to 4-HBA and CoA occurred with a k cat of 6.7 s−1 and a Km of 1.2 μM. The k cat pH rate profile for 4-HBA-CoA hydrolysis indicated optimal activity over a pH range of 6 to 10. The amino acid sequence of the FcbC protein was compared to other sequences contained in the protein sequence data banks. A large number of sequence homologues of unknown function were identified. On the other hand, the 4-HBA-CoA thioesterases isolated from 4-CBA-degrading Pseudomonas strains did not share significant sequence identity with the FcbC protein, indicating early divergence of the thioesterase-encoding genes.

scholarly journals Characterization of Benzoyl Coenzyme A Biosynthesis Genes in the Enterocin-Producing Bacterium “Streptomyces maritimus”

2003 ◽  
Vol 185 (2) ◽  
pp. 399-404 ◽  
Author(s):  
Longkuan Xiang ◽  
Bradley S. Moore
Keyword(s):  
Benzoic Acid ◽  
Phenylalanine Ammonia Lyase ◽  
Coenzyme A ◽  
Primary Metabolism ◽  
The Novel ◽  
Wild Type ◽  
Coa Ligase ◽  
Encoding Gene ◽  
Coenzyme A Biosynthesis

ABSTRACT The novel benzoyl coenzyme A (benzoyl-CoA) biosynthesis pathway in “Streptomyces maritimus” was investigated through a series of target-directed mutations. Genes involved in benzoyl-CoA formation were disrupted through single-crossover homologous recombination, and the resulting mutants were analyzed for their ability to biosynthesize the benzoyl-CoA-primed polyketide antibiotic enterocin. Inactivation of the unique phenylalanine ammonia-lyase-encoding gene encP was previously shown to be absolutely required for benzoyl-CoA formation in “S. maritimus”. The fatty acid β-oxidation-related genes encH, -I, and -J, on the other hand, are necessary but not required. In each case, the yield of benzoyl-CoA-primed enterocin dropped below wild-type levels. We attribute the reduced benzoyl-CoA formation in these specific mutants to functional substitution and cross-talk between the products of genes encH, -I, and -J and the enzyme homologues of primary metabolism. Disruption of the benzoate-CoA ligase encN gene did not perturb enterocin production, however, demonstrating that encN is extraneous and that benzoic acid is not a pathway intermediate. EncN rather serves as a substitute pathway for utilizing exogenous benzoic acid. These experiments provide further support that benzoyl-CoA is formed in a novel bacterial pathway that resembles the eukaryotic assembly of benzoyl-CoA from phenylalanine via a β-oxidative path.

scholarly journals Characterization of Neurospora crassa catabolic dehydroquinase purified from N. crassa and Escherichia coli

1982 ◽  
Vol 203 (3) ◽  
pp. 769-773 ◽  
Author(s):  
A R Hawkins ◽  
W R Reinert ◽  
N H Giles
Keyword(s):  
Escherichia Coli ◽  
Steady State ◽  
Neurospora Crassa ◽  
Protein Sequence ◽  
Sequence Data ◽  
E Coli ◽  
Steady State Kinetics ◽  
Electrophoretic Data ◽  
Protein Sequence Data

1. Neurospora crassa catabolic dehydroquinase has been purified from N. crassa and Escherichia coli. 2. Protein-sequence and gel-electrophoretic data show that apparently pure, homogeneous native dehydroquinase is a mixture of intact and proteinase-cleaved enzyme monomers. 3. Protein-sequence data and steady-state kinetics show that the catabolic dehydroquinase gene of N. crassa is expressed with fidelity in E. coli.

scholarly journals Novel Characteristics of Succinate Coenzyme A (Succinate-CoA) Ligases: Conversion of Malate to Malyl-CoA and CoA-Thioester Formation of Succinate AnaloguesIn Vitro

2013 ◽  
Vol 80 (1) ◽  
pp. 166-176 ◽  
Author(s):  
Johannes Christoph Nolte ◽  
Marc Schürmann ◽  
Catherine-Louise Schepers ◽  
Elvira Vogel ◽  
Jan Hendrik Wübbeler ◽  
...  
Keyword(s):  
Coenzyme A ◽  
Degradation Pathway ◽  
Ionization Mass ◽  
Content Type ◽  
E Coli ◽  
Coa Ligase ◽  
Strong Resemblance ◽  
Coa Thioesters

ABSTRACTThree succinate coenzyme A (succinate-CoA) ligases (SucCD) fromEscherichia coli,Advenella mimigardefordensisDPN7T, andAlcanivorax borkumensisSK2 were characterized regarding their substrate specificity concerning succinate analogues. Previous studies had suggested that SucCD enzymes might be promiscuous toward succinate analogues, such as itaconate and 3-sulfinopropionate (3SP). The latter is an intermediate of the degradation pathway of 3,3′-dithiodipropionate (DTDP), a precursor for the biotechnical production of polythioesters (PTEs) in bacteria. ThesucCDgenes were expressed inE. coliBL21(DE3)/pLysS. The SucCD enzymes ofE. coliandA. mimigardefordensisDPN7Twere purified in the native state using stepwise purification protocols, while SucCD fromA. borkumensisSK2 was equipped with a C-terminal hexahistidine tag at the SucD subunit. Besides the preference for the physiological substrates succinate, itaconate, ATP, and CoA, high enzyme activity was additionally determined for both enantiomeric forms of malate, amounting to 10 to 21% of the activity with succinate.Kmvalues ranged from 2.5 to 3.6 mM forl-malate and from 3.6 to 4.2 mM ford-malate for the SucCD enzymes investigated in this study. Asl-malate-CoA ligase is present in the serine cycle for assimilation of C1compounds in methylotrophs, structural comparison of these two enzymes as members of the same subsubclass suggested a strong resemblance of SucCD tol-malate-CoA ligase and gave rise to the speculation that malate-CoA ligases and succinate-CoA ligases have the same evolutionary origin. Although enzyme activities were very low for the additional substrates investigated, liquid chromatography/electrospray ionization-mass spectrometry analyses proved the ability of SucCD enzymes to form CoA-thioesters of adipate, glutarate, and fumarate. Since all SucCD enzymes were able to activate 3SP to 3SP-CoA, we consequently demonstrated that the activation of 3SP is not a unique characteristic of the SucCD fromA. mimigardefordensisDPN7T. The essential role ofsucCDin the activation of 3SPin vivowas proved by genetic complementation.

scholarly journals Purification and partial characterization of four proteins from human parotid saliva

1971 ◽  
Vol 123 (3) ◽  
pp. 455-464 ◽  
Author(s):  
Anders Bennick ◽  
George E. Connell
Keyword(s):  
Serum Proteins ◽  
Protein A ◽  
Gel Filtration ◽  
Binding Activity ◽  
Significant Activity ◽  
Parotid Saliva ◽  
Polyacrylamide Electrophoresis ◽  
Ph Range ◽  
Human Parotid Saliva

Four proteins, which have been designated A, B, C and D, have been purified from human parotid saliva. These proteins are the major constituents of parotid saliva which migrate rapidly to the anode in polyacrylamide electrophoresis at pH9.5. Gel filtration and polyacrylamide electrophoresis were employed in the purification procedures. After purification all four preparations were tested for homogeneity by electrophoresis at pH2.8 and 9.5, by isoelectric focusing in the pH range 3–10, by immunodiffusion, and by sedimentation in the analytical ultracentrifuge. None of the proteins showed significant activity in assays for amylase, acid and alkaline phosphatase, protease, lysozyme, ribonuclease, peroxidase, β-glucuronidase, β-galactosidase, iron-binding activity and esterase. No cross-reactions were detected with antisera specific for lactoferrin and 15 serum proteins. All four proteins were rich in glutamic acid, proline and glycine and were lacking completely the sulphur-containing amino acids. Proteins A and C contained no threonine or tyrosine. Carbohydrate could be demonstrated only in protein A at a concentration of 4% of the total protein.

scholarly journals Integration of Residue Attributes for Sequence Diversity Characterization of Terpenoid Enzymes

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Nelson Kibinge ◽  
Shun Ikeda ◽  
Naoaki Ono ◽  
Md. Altaf-Ul-Amin ◽  
Shigehiko Kanaya
Keyword(s):  
Amino Acid ◽  
Sequence Data ◽  
Principal Component ◽  
Analytical Techniques ◽  
Molecular Data ◽  
Amino Acid Index ◽  
Protein Sequence Data ◽  
Physical Attributes ◽  
Acid Index

Progress in the “omics” fields such as genomics, transcriptomics, proteomics, and metabolomics has engendered a need for innovative analytical techniques to derive meaningful information from the ever increasing molecular data. KNApSAcK motorcycle DB is a popular database for enzymes related to secondary metabolic pathways in plants. One of the challenges in analyses of protein sequence data in such repositories is the standard notation of sequences as strings of alphabetical characters. This has created lack of a natural underlying metric that eases amenability to computation. In view of this requirement, we applied novel integration of selected biochemical and physical attributes of amino acids derived from the amino acid index and quantified in numerical scale, to examine diversity of peptide sequences of terpenoid synthases accumulated in KNApSAcK motorcycle DB. We initially generated a reduced amino acid index table. This is a set of biochemical and physical properties obtained by random forest feature selection of important indices from the amino acid index. Principal component analysis was then applied for characterization of enzymes involved in synthesis of terpenoids. The variance explained was increased by incorporation of residue attributes for analyses.

Isolation and Characterization of Plasminogen Activator from Pig Leucocytes

1974 ◽  
Vol 31 (01) ◽  
pp. 072-085 ◽  
Author(s):  
M Kopitar ◽  
M Stegnar ◽  
B Accetto ◽  
D Lebez
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Isolation And Characterization ◽  
Ph Range ◽  
Inhibition Studies ◽  
Final Purification

SummaryPlasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

scholarly journals Analysis of the Mechanism by Which Glucose Inhibits Maltose Induction of MAL Gene Expression in Saccharomyces

Genetics ◽  
2000 ◽  
Vol 154 (1) ◽  
pp. 121-132
Author(s):  
Zhen Hu ◽  
Yingzi Yue ◽  
Hua Jiang ◽  
Bin Zhang ◽  
Peter W Sherwood ◽  
...  
Keyword(s):  
Gene Expression ◽  
Glucose Repression ◽  
Protein A ◽  
The Other ◽  
Maltose Permease ◽  
Inducer Exclusion ◽  
Glucose Inhibition ◽  
Independent Mechanism ◽  
Mal Genes

Abstract Expression of the MAL genes required for maltose fermentation in Saccharomyces cerevisiae is induced by maltose and repressed by glucose. Maltose-inducible regulation requires maltose permease and the MAL-activator protein, a DNA-binding transcription factor encoded by MAL63 and its homologues at the other MAL loci. Previously, we showed that the Mig1 repressor mediates glucose repression of MAL gene expression. Glucose also blocks MAL-activator-mediated maltose induction through a Mig1p-independent mechanism that we refer to as glucose inhibition. Here we report the characterization of this process. Our results indicate that glucose inhibition is also Mig2p independent. Moreover, we show that neither overexpression of the MAL-activator nor elimination of inducer exclusion is sufficient to relieve glucose inhibition, suggesting that glucose acts to inhibit induction by affecting maltose sensing and/or signaling. The glucose inhibition pathway requires HXK2, REG1, and GSF1 and appears to overlap upstream with the glucose repression pathway. The likely target of glucose inhibition is Snf1 protein kinase. Evidence is presented indicating that, in addition to its role in the inactivation of Mig1p, Snf1p is required post-transcriptionally for the synthesis of maltose permease whose function is essential for maltose induction.

Characterization of Nucleotide Binding ­Site-Encoding Genes in Sweetpotato, Ipomoea batatas(L.) Lam., and Their Response to Biotic and Abiotic Stresses

2021 ◽  
pp. 1-15
Author(s):  
Zengzhi Si ◽  
Yake Qiao ◽  
Kai Zhang ◽  
Zhixin Ji ◽  
Jinling Han
Keyword(s):  
Binding Site ◽  
Abiotic Stresses ◽  
Ipomoea Batatas ◽  
Expression Profiles ◽  
Nucleotide Binding ◽  
Regulatory Elements ◽  
Nucleotide Binding Site ◽  
Biotic And Abiotic Stresses ◽  
Encoding Genes

Sweetpotato, <i>Ipomoea batatas</i> (L.) Lam., is an important and widely grown crop, yet its production is affected severely by biotic and abiotic stresses. The nucleotide binding site (NBS)-encoding genes have been shown to improve stress tolerance in several plant species. However, the characterization of NBS-encoding genes in sweetpotato is not well-documented to date. In this study, a comprehensive analysis of NBS-encoding genes has been conducted on this species by using bioinformatics and molecular biology methods. A total of 315 NBS-encoding genes were identified, and 260 of them contained all essential conserved domains while 55 genes were truncated. Based on domain architectures, the 260 NBS-encoding genes were grouped into 6 distinct categories. Phylogenetic analysis grouped these genes into 3 classes: TIR, CC (I), and CC (II). Chromosome location analysis revealed that the distribution of NBS-encoding genes in chromosomes was uneven, with a number ranging from 1 to 34. Multiple stress-related regulatory elements were detected in the promoters, and the NBS-encoding genes’ expression profiles under biotic and abiotic stresses were obtained. According to the bioinformatics analysis, 9 genes were selected for RT-qPCR analysis. The results revealed that <i>IbNBS75</i>, <i>IbNBS219</i>, and <i>IbNBS256</i> respond to stem nematode infection; <i>Ib­NBS240</i>, <i>IbNBS90</i>, and <i>IbNBS80</i> respond to cold stress, while <i>IbNBS208</i>, <i>IbNBS71</i>, and <i>IbNBS159</i> respond to 30% PEG treatment. We hope these results will provide new insights into the evolution of NBS-encoding genes in the sweetpotato genome and contribute to the molecular breeding of sweetpotato in the future.

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