Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 4 de 4
Filter
Add more filters










Database
Language
Publication year range
1.
Mol Cell Biochem ; 400(1-2): 9-15, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25355158

ABSTRACT

Recently, we showed that the fused chorismate-utilizing enzyme from the antibiotic-producing soil bacterium Streptomyces venezuelae is an anthranilate synthase (designated SvAS), not a 2-amino-2-deoxyisochorismate (ADIC) synthase, as was predicted based on its amino acid sequence similarity to the phenazine biosynthetic enzyme PhzE (an ADIC synthase). Here, we report the characterization of SvAS using steady-state kinetics, gel filtration chromatography, and laser light scattering. The recombinant His-tagged enzyme has Michaelis constants Km with respect to substrates chorismate and glutamine of 8.2 ± 0.2 µM and 0.84 ± 0.05 mM, respectively, and a catalytic rate constant k cat of 0.57 ± 0.02 s(-1) at 30 °C. Unlike most other anthranilate synthases, SvAS does not utilize ammonia as a substrate. The enzyme is competitively but non-cooperatively inhibited by tryptophan (K i = 11.1 ± 0.1 µM) and is active as a monomer. The finding that SvAS is a monomer jibes with the variety of association modes that have been observed for anthranilate synthases from different microorganisms, and it identifies the enzyme's minimal functional unit as a single TrpE-TrpG pair.


Subject(s)
Anthranilate Synthase/chemistry , Catalysis , Streptomyces/enzymology , Amino Acid Sequence/genetics , Anthranilate Synthase/genetics , Kinetics , Protein Structure, Tertiary , Substrate Specificity , Tryptophan
2.
Mol Cell Biochem ; 387(1-2): 207-16, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24248535

ABSTRACT

As part of an overall project to characterize the streptomycin phosphotransferase enzyme APH(6)-Id, which confers bacterial resistance to streptomycin, we cloned, expressed, purified, and characterized the enzyme. When expressed in Escherichia coli, the recombinant enzyme increased by up to 70-fold the minimum inhibitory concentration needed to inhibit cell growth. Size-exclusion chromatography gave a molecular mass of 31.4 ± 1.3 kDa for the enzyme, showing that it functions as a monomer. Activity was assayed using three methods: (1) an HPLC-based method that measures the consumption of streptomycin over time; (2) a spectrophotometric method that utilizes a coupled assay; and (3) a radioenzymatic method that detects production of (32)P-labeled streptomycin phosphate. Altogether, the three methods demonstrated that streptomycin was consumed in the APH(6)-Id-catalyzed reaction, ATP was hydrolyzed, and streptomycin phosphate was produced in a substrate-dependent manner, demonstrating that APH(6)-Id is a streptomycin phosphotransferase. Steady-state kinetic analysis gave the following results: K(m)(streptomycin) of 0.38 ± 0.13 mM, K(m)(ATP) of 1.03 ± 0.1 mM, V(max) of 3.2 ± 1.1 µmol/min/mg, and k(cat) of 1.7 ± 0.6 s(-1). Our study demonstrates that APH(6)-Id is a bona fide streptomycin phosphotransferase, functions as a monomer, and confers resistance to streptomycin.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/chemistry , Phosphotransferases (Alcohol Group Acceptor)/chemistry , Streptomycin/pharmacology , Anti-Bacterial Agents/chemistry , Bacterial Proteins/biosynthesis , Bacterial Proteins/isolation & purification , Chromatography, High Pressure Liquid , Drug Resistance, Bacterial , Escherichia coli/drug effects , Escherichia coli/metabolism , Kinetics , Microbial Sensitivity Tests , Phosphotransferases (Alcohol Group Acceptor)/biosynthesis , Phosphotransferases (Alcohol Group Acceptor)/isolation & purification , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Streptomyces/enzymology , Streptomycin/chemistry
3.
Article in English | MEDLINE | ID: mdl-22954966

ABSTRACT

In the current study, we developed a HPLC method to quantitatively measure the permeability of the BpT-based chelators, 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT) and 2-benzoylpyridine 4-allyl-3-thiosemicarbazone (Bp4aT), across human colorectal adenocarcinoma (Caco-2) monolayers as a model of gut absorption. In aqueous solution, Bp4eT and Bp4aT formed inter-convertible Z and E isomers that were resolved by HPLC. Peak area was linear with respect to chelator concentration. Acceptable within-day and between-day precision (<22%) and accuracy (85-115% of true values) were obtained over a range of 1.0-100µM for Bp4eT and 1.5-300µM for Bp4aT. Limits of detection were 0.3µM and 1µM for Bp4eT and Bp4aT, respectively, while corresponding limits of quantification were 1µM and 5µM. Both chelators showed significant ability to chelate iron in THP-1 cells using a calcein-based assay and no apparent cytotoxicity was observed within 24h. Ratios of the apical to basolateral and basolateral to apical transport for Bp4eT were 1.10 and 0.89 at 100µM and 300µM respectively, indicating equal bi-directional movement of the compounds. Similarly, ratios were 0.77 and 0.92 for Bp4aT, respectively. This study demonstrates that Bp4eT and Bp4aT can be efficiently transported through Caco-2 cells and can potentially be formulated for oral delivery.


Subject(s)
Chromatography, High Pressure Liquid/methods , Iron Chelating Agents/analysis , Iron Chelating Agents/pharmacokinetics , Thiosemicarbazones/analysis , Thiosemicarbazones/pharmacokinetics , Caco-2 Cells , Cell Line, Tumor , Cell Membrane Permeability , Cell Survival/drug effects , Humans , Iron/metabolism , Iron Chelating Agents/chemistry , Iron Chelating Agents/pharmacology , Isomerism , Isoquinolines/metabolism , Limit of Detection , Models, Biological , Reproducibility of Results , Thiosemicarbazones/chemistry , Thiosemicarbazones/pharmacology
4.
Ethn Dis ; 18(2 Suppl 2): S2-9-13, 2008.
Article in English | MEDLINE | ID: mdl-18646313

ABSTRACT

The chloramphenicol producer Streptomyces venezuelae contains an enzyme, SvTrpEG, that has a high degree of amino acid sequence similarity to the phenazine biosynthetic enzyme PhzE of certain species of Pseudomonas. PhzE has the sequence signature of an anthranilate synthase, but recent evidence indicates that it catalyzes the production of 2-amino-2-deoxyisochorismate [corrected] (ADIC), an intermediate in the two-step anthranilate synthase reaction, not anthranilate. In order to determine if SvTrpEG is likewise an ADIC synthase, we have cloned the gene for SvTrpEG, expressed the recombinant enzyme in Escherichia coli, and purified the enzyme. Analysis of the SvTrpEG-catalyzed reaction mixture using UV-visible spectrophotometry, fluorescence spectrometry, and high-performance liquid chromatography shows that the product of the reaction is anthranilate, not ADIC. Our results therefore reveal that, despite its sequence similarity to PhzE, SvTrpEG is an anthranilate synthase, not an ADIC synthase.


Subject(s)
Anthranilate Synthase/genetics , Genes, Bacterial/genetics , Streptomyces/enzymology , Streptomyces/genetics , Anthranilate Synthase/metabolism , Base Sequence , Catalysis , Chorismic Acid/metabolism , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Escherichia coli/enzymology , Escherichia coli/genetics , Kinetics , Molecular Sequence Data , Plasmids/genetics , Polymerase Chain Reaction , Pseudomonas/enzymology , Pseudomonas/genetics , Salmonella/enzymology , Salmonella/genetics , Thermodynamics , Transformation, Genetic
SELECTION OF CITATIONS
SEARCH DETAIL
...