(De)regulation of key enzyme steps in the shikimate pathway and phenylalanine-specific pathway of the actinomycete Amycolatopsis methanolica

H. Kloosterman, G.I. Hessels, J.W. Vrijbloed, G.J. Euverink, L. Dijkhuizen

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22 Citations (Scopus)

Abstract

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, feed back-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.

Original languageEnglish
Pages (from-to)3321-3330
Number of pages10
JournalMicrobiology-Sgm
Volume149
Issue number11
DOIs
Publication statusPublished - Nov-2003

Keywords

  • 3-DEOXY-D-ARABINO-HEPTULOSONATE 7-PHOSPHATE SYNTHASE
  • MUTASE-PREPHENATE DEHYDRATASE
  • STREPTOMYCES-COELICOLOR A3(2)
  • COMPLETE GENOME SEQUENCE
  • AMINO-ACID BIOSYNTHESIS
  • ESCHERICHIA-COLI K-12
  • NOCARDIA SP 239
  • CHORISMATE MUTASE
  • 3-AMINO-5-HYDROXYBENZOIC ACID
  • ANSAMYCIN ANTIBIOTICS

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