(De)regulation of key enzyme steps in the shikimate pathway and phenylalanine-specific pathway of the actinomycete Amycolatopsis methanolica.
Prephenate dehydratase (PDT), chorismate mutase (CM) and 3-deoxy-D-arabino-7-heptulosonate 7-phosphate (DAHP) synthase are key regulatory enzymes in aromatic amino acid biosynthesis in the actinomycete Amycolatopsis methanolica. Deregulated, feedback-control-resistant mutants were isolated by incubation of A. methanolica on glucose mineral agar containing the toxic analogue p-fluoro-DL-phenylalanine (pFPhe). Several of these mutants had completely lost PDT sensitivity to Phe inhibition and Tyr activation. Mutant characterization yielded new information about PDT amino acid residues involved in Phe and Tyr effector binding sites. A. methanolica wild-type cells grown on glucose mineral medium normally possess a bifunctional CM/DAHP synthase protein complex (with DS1, a plant-type DAHP synthase). The CM activity of this protein complex is feedback-inhibited by Tyr and Phe, while DS1 activity is mainly inhibited by Trp. Isolation of pFPhe-resistant mutants yielded two feedback-inhibition-resistant CM mutants. These were characterized as regulatory mutants, derepressed in (a) synthesis of CM, now occurring as an abundant, feedback-inhibition-resistant, separate protein, and (b) synthesis of an alternative DAHP synthase (DS2, an E. coli-type DAHP synthase), only inhibited by Tyr and Trp. DS1 and DS2 thus are well integrated in A. methanolica primary metabolism: DS1 and CM form a protein complex, which stimulates CM activity and renders it sensitive to feedback inhibition by Phe and Tyr. Synthesis of CM and DS2 proteins appears to be controlled co-ordinately, sensitive to Phe-mediated feedback repression.
Kloosterman
H
H
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, PO Box 14, 9750 AA Haren, The Netherlands.
Hessels
G I
GI
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, PO Box 14, 9750 AA Haren, The Netherlands.
Vrijbloed
J W
JW
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, PO Box 14, 9750 AA Haren, The Netherlands.
Euverink
G J
GJ
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, PO Box 14, 9750 AA Haren, The Netherlands.
Dijkhuizen
L
L
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, PO Box 14, 9750 AA Haren, The Netherlands.
D010723:3-Deoxy-7-Phosphoheptulonate Synthase / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D000190:Actinomyces / Q000201:enzymology*; D000595:Amino Acid Sequence; D049148:Aminoacylation; D002826:Chorismate Mutase / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000378:metabolism / Q000494:pharmacology; D011302:Prephenate Dehydratase / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012765:Shikimic Acid / Q000378:metabolism; D014443:Tyrosine / Q000494:pharmacology
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D012765:Shikimic Acid; D014443:Tyrosine; D010649:Phenylalanine; D010723:3-Deoxy-7-Phosphoheptulonate Synthase; D011302:Prephenate Dehydratase; D002826:Chorismate Mutase