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1.
Biotechnol Prog ; 22(4): 1003-11, 2006.
Article in English | MEDLINE | ID: mdl-16889377

ABSTRACT

The incorporation of stable isotopes in suspension cultured cells is very simple and useful as a preliminary experimental method in the experimental scene of plant metabolomics to elucidate the metabolic profiles of mutants and transformants. Stable isotope methods would afford a dynamic explanation of turnover speed that would concern the metabolic flux. Utilization of suspension cultured cells allows genes to be easily induced or suppressed, culture conditions to be controlled, and samples to be easily prepared. Stable isotope tracing allows an index of metabolic flux to be obtained. Here we present an experiment feeding (15)N-labeled inorganic salts to Arabidopsis (cell line T87) and Coptis cultured cells. Results of a comparison of (15)N labeling ratios of amino acids derived from T87 cells cultured under light with those cultured in the dark corresponded to transcriptional expressions revealed by microarray experiments published previously, demonstrating the validity of this procedure. Furthermore, (15)N labeling ratios of Coptis cultured cells revealed arginine and lysine metabolism inhibition, which should result in inhibition of polyamine biosynthesis and cell division. This very simple experiment allowed us to uncover metabolic dynamic features of the plant cell. Therefore this method is very useful for forming working hypotheses and experimental design.


Subject(s)
Arabidopsis/metabolism , Coptis/metabolism , Nitrates/metabolism , Quaternary Ammonium Compounds/metabolism , Amino Acids/antagonists & inhibitors , Amino Acids/chemistry , Amino Acids/metabolism , Arabidopsis/chemistry , Arabidopsis/cytology , Cell Culture Techniques/methods , Cell Division , Cell Line , Chorismate Mutase/analysis , Chorismate Mutase/biosynthesis , Chorismate Mutase/genetics , Coptis/chemistry , Coptis/cytology , Mass Spectrometry/methods , Nitrates/chemistry , Nitrates/pharmacology , Nitrogen Isotopes , Protein Array Analysis/methods , Quaternary Ammonium Compounds/chemistry , Quaternary Ammonium Compounds/pharmacology , Sensitivity and Specificity
2.
Bioorg Med Chem ; 11(14): 3109-14, 2003 Jul 17.
Article in English | MEDLINE | ID: mdl-12818673

ABSTRACT

Besides playing a central role in phenylalanine biosynthesis, the bifunctional P-protein in Eschericia coli provides a unique model system for investigating whether allosteric effects can be engineered into protein catalysts using modular regulatory elements. Previous studies have established that the P-protein contains three distinct domains whose functions are preserved, even when separated: chorismate mutase (residues 1-109), prephenate dehydratase (residues 101-285), and an allosteric domain (residues 286-386) for feedback inhibition by phenylalanine. By deleting the prephenate dehydrase domain, a functional chorismate mutase linked directly to the phenylalanine binding domain has been engineered and overexpressed. This manuscript reports the catalytic properties of the mutase in the absence and presence of phenylalanine.


Subject(s)
Allosteric Regulation/genetics , Chorismate Mutase/genetics , Escherichia coli Proteins/genetics , Multienzyme Complexes/genetics , Prephenate Dehydratase/genetics , Chorismate Mutase/biosynthesis , Chorismate Mutase/chemistry , Chorismate Mutase/metabolism , Escherichia coli Proteins/biosynthesis , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Kinetics , Multienzyme Complexes/biosynthesis , Multienzyme Complexes/chemistry , Multienzyme Complexes/metabolism , Phenylalanine , Prephenate Dehydratase/biosynthesis , Prephenate Dehydratase/chemistry , Prephenate Dehydratase/metabolism , Protein Engineering
3.
J Bacteriol ; 182(15): 4188-97, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10894726

ABSTRACT

The HARO7 gene of the methylotrophic, thermotolerant yeast Hansenula polymorpha was cloned by functional complementation. HARO7 encodes a monofunctional 280-amino-acid protein with chorismate mutase (EC 5.4. 99.5) activity that catalyzes the conversion of chorismate to prephenate, a key step in the biosynthesis of aromatic amino acids. The HARO7 gene product shows strong similarities to primary sequences of known eukaryotic chorismate mutase enzymes. After homologous overexpression and purification of the 32-kDa protein, its kinetic parameters (k(cat) = 319.1 s(-1), n(H) = 1.56, [S](0.5) = 16.7 mM) as well as its allosteric regulatory properties were determined. Tryptophan acts as heterotropic positive effector; tyrosine is a negative-acting, heterotropic feedback inhibitor of enzyme activity. The influence of temperature on catalytic turnover and the thermal stability of the enzyme were determined and compared to features of the chorismate mutase enzyme of Saccharomyces cerevisiae. Using the Cre-loxP recombination system, we constructed mutant strains carrying a disrupted HARO7 gene that showed tyrosine auxotrophy and severe growth defects. The amount of the 0.9-kb HARO7 mRNA is independent of amino acid starvation conditions but increases twofold in the presence of methanol as the sole carbon source, implying a catabolite repression system acting on HARO7 expression.


Subject(s)
Chorismate Mutase/biosynthesis , Chorismate Mutase/genetics , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Fungal , Methanol/metabolism , Pichia/enzymology , Amino Acid Sequence , Catalysis , Cyclohexanecarboxylic Acids/metabolism , Cyclohexenes , Kinetics , Molecular Sequence Data , Saccharomyces cerevisiae/enzymology , Temperature
4.
Biochemistry ; 38(38): 12212-7, 1999 Sep 21.
Article in English | MEDLINE | ID: mdl-10493788

ABSTRACT

Isothermal titration calorimetry (ITC) and site-directed mutagenesis were used to study the interaction of Phe with (a) the Escherichia coli P-protein, a bifunctional chorismate mutase/prephenate dehydratase that is feedback inhibited by Phe, (b) PDT32, a 32 kDa P-protein fragment (residues 101-386) containing the prephenate dehydratase and regulatory domains, and (c) R12, a C-terminal 12 kDa P-protein fragment (residues 286-386) containing the regulatory domain. DeltaH(total) values for PDT32, which included the heats of Phe binding, conformational change, and dimerization, established that in developing a mechanism for end product feedback inhibition, the P-protein has evolved a ligand recognition domain that exhibits Phe-binding enthalpies comparable to those reported for other full-fledged amino acid receptor proteins. Sequence alignments of R12 with other Phe-binding enzymes identified two highly conserved regions, GALV (residues 309-312) and ESRP (residues 329-332). Site-directed mutagenesis and ITC established that changes in the GALV and ESRP regions affected Phe binding and feedback inhibition to different extents. Mutagenesis further showed that C374 was essential for feedback inhibition, but not for Phe binding, while W338 was involved in Phe binding, but not in the Phe-induced conformational change required for feedback inhibition.


Subject(s)
Bacterial Proteins/antagonists & inhibitors , Chorismate Mutase/antagonists & inhibitors , Escherichia coli Proteins , Escherichia coli/enzymology , Multienzyme Complexes/antagonists & inhibitors , Phenylalanine/biosynthesis , Phenylalanine/metabolism , Prephenate Dehydratase/antagonists & inhibitors , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites/genetics , Calorimetry , Chorismate Mutase/biosynthesis , Chorismate Mutase/genetics , Chorismate Mutase/metabolism , Chromatography, Gel , Feedback , Multienzyme Complexes/biosynthesis , Multienzyme Complexes/genetics , Multienzyme Complexes/metabolism , Mutagenesis, Site-Directed , Peptide Fragments/biosynthesis , Peptide Fragments/genetics , Peptide Fragments/metabolism , Phenylalanine/genetics , Prephenate Dehydratase/biosynthesis , Prephenate Dehydratase/genetics , Prephenate Dehydratase/metabolism , Protein Binding/genetics , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
5.
J Biol Chem ; 274(32): 22275-82, 1999 Aug 06.
Article in English | MEDLINE | ID: mdl-10428795

ABSTRACT

The cDNA and the chromosomal locus of the aroC gene of Aspergillus nidulans were cloned and is the first representative of a filamentous fungal gene encoding chorismate mutase (EC 5.4.99.5), the enzyme at the first branch point of aromatic amino acid biosynthesis. The aroC gene complements the Saccharomyces cerevisiae aro7Delta as well as the A. nidulans aroC mutation. The gene consists of three exons interrupted by two short intron sequences. The expressed mRNA is 0.96 kilobases in length and aroC expression is not regulated on the transcriptional level under amino acid starvation conditions. aroC encodes a monofunctional polypeptide of 268 amino acids. Purification of this 30-kDa enzyme allowed determination of its kinetic parameters (k(cat) = 82 s(-1), n(H) = 1. 56, [S](0.5) = 2.3 mM), varying pH dependence of catalytic activity in different regulatory states, and an acidic pI value of 4.7. Tryptophan acts as heterotropic activator and tyrosine as negative acting, heterotropic feedback-inhibitor with a K(i) of 2.8 microM. Immunological data, homology modeling, as well as electron microscopy studies, indicate that this chorismate mutase has a dimeric structure like the S. cerevisiae enzyme. Site-directed mutagenesis of a crucial residue in loop220s (Asp(233)) revealed differences concerning the intramolecular signal transduction for allosteric regulation of enzymatic activity.


Subject(s)
Aspergillus nidulans/genetics , Chorismate Mutase/genetics , Genes, Fungal , Phosphorus-Oxygen Lyases/genetics , Allosteric Regulation , Amino Acid Sequence , Aspartic Acid/genetics , Aspergillus nidulans/enzymology , Chorismate Mutase/biosynthesis , Chorismate Mutase/drug effects , Chorismate Mutase/ultrastructure , Chorismic Acid/metabolism , Computer Simulation , Hydrogen-Ion Concentration , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Phosphorus-Oxygen Lyases/biosynthesis , Phosphorus-Oxygen Lyases/drug effects , Phosphorus-Oxygen Lyases/ultrastructure , Protein Conformation , Recombinant Proteins/biosynthesis , Sequence Homology, Amino Acid , Tryptophan/metabolism , Tyrosine/pharmacology
6.
FEBS Lett ; 334(2): 233-6, 1993 Nov 15.
Article in English | MEDLINE | ID: mdl-8224252

ABSTRACT

Chorismate mutase (EC 5.4.99.5) catalyzes the first step in the branch of the shikimate pathway which leads to the aromatic amino acids, phenylalanine and tyrosine. We have isolated a cDNA for this enzyme from the higher plant, Arabidopsis thaliana, by complementing a yeast strain (aro7) with a cDNA library from A. thaliana. This is the first chorismate mutase cDNA isolated from a plant. It encodes a protein of 334 amino acids. The identity of the deduced amino acid sequence is 41% to the chorismate mutase sequence from Saccharomyces cerevisiae. The N-terminal portion of the deduced amino acid sequence has no homology to the S. cerevisiae sequence but resembles known plastid-specific transit peptides. The A. thaliana chorismate mutase expressed in yeast revealed allosteric control by the three aromatic amino acids, as previously described for plastidic chorismate mutase isozymes.


Subject(s)
Arabidopsis/enzymology , Chorismate Mutase/biosynthesis , Chorismate Mutase/genetics , DNA, Complementary/chemistry , Saccharomyces cerevisiae/enzymology , Amino Acid Sequence , Arabidopsis/genetics , Base Sequence , Chorismate Mutase/metabolism , Cloning, Molecular/methods , Gene Expression , Kinetics , Molecular Sequence Data , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Sequence Homology, Amino Acid
7.
Acta Microbiol Acad Sci Hung ; 23(2): 167-70, 1976.
Article in English | MEDLINE | ID: mdl-9783

ABSTRACT

Several types of 4-fluorophenylalanine resistant mutants were isolated. In one type of mutant DAHP synthetase (tyr) and prephenate dehydrogenase were coordinately derepressed. The mutation was linked to aroF and tyrA and was cis- dominant by merodiploid analysis, thus confirming that it is an operator constitutive mutation (tyrOc). A second type of mutation showed highly elevated levels of tyrosine pathway enzymes which were not repressed by L-tyrosine. It was unlinked to tyrA and aroF, and was trans-recessive in merodiploids. These properties were attributed to a mutation in a regulator gene, tyrR (linked to pyr F), that resulted in altered or non-functional aporepressor. Hence tyrO, tyrA, and aroF constitute an operon regulated by tyrR. In a third type of mutation chorismate mutase P-prephenate dehydratase was highly elevated. It was not linked to pheA, was located in the 95--100 min region of the Salmonella chromosome, and was recessive to the wild type gene in merodiploids. A mutation was, therefore, indicated in a regulatory gene, pheR, which specified an aporepressor for regulating pheA. DAHP synthetase (phe), specified by aroG, was not regulated by pheR, but was derepressed in one of the tyrR mutants, suggesting that as in Escherichia coli tyrR may regulate DAHP synthetase(phe) and DAHP synthetase (tyr) with the same aporepressor. A novel mutation in chorismate mutase is described.


Subject(s)
Genes, Regulator , Phenylalanine/biosynthesis , Salmonella typhimurium/metabolism , Tyrosine/biosynthesis , 3-Deoxy-7-Phosphoheptulonate Synthase/biosynthesis , Chorismate Mutase/biosynthesis , Drug Resistance, Microbial , Enzyme Repression , Genetic Linkage , Mutation , Prephenate Dehydratase/biosynthesis , Prephenate Dehydrogenase/biosynthesis , Salmonella typhimurium/drug effects , Salmonella typhimurium/enzymology , p-Fluorophenylalanine/pharmacology
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