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

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, 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. INTRODUCTIONIn micro-organisms and plants the biosynthesis of aromatic compounds proceeds via the common seven-step aromatic or shikimate pathway to the branch point intermediate chorismate. This intermediate is subsequently converted to the three aromatic amino acids via specific terminal pathways (Fig. 1). Many other (aromatic) compounds are derived either partially or entirely from chorismate or from other pathway intermediates or end products, e.g. pyrroloquinoline quinone, lignin, ubiquinone, plastiquinone, enterochelin, vitamin K and 3-amino-5-hydroxybenzoic acid, the precursor of the mC 7 N units found in mitomycin and ansamycin antibiotics (Bentley, 1990;Knaggs, 1999;Arakawa et al., 2002). Aromatic amino acid biosynthesis in bacteria is strictly regulated via feedback control mechanisms. Limited information is available about these enzymes or their regulation in actinomycetes, Gram-positive bacteria that are well-known producers of numerous antibiotics derived from aromatic amino acids or their pathway intermediates (Hodgson, 2000). Antibiotic biosynthesis may require specific metabolic adaptations, e.g. expression of isoenzymes that serve to avoid feedback regulation by aromatic amino acids.We are interested in the enzymology and regulation of aromatic amino acid biosynthesis in the nocardioform actinomycete Amycolatopsis methanolica. This bacterium is closely related to Amycolatopsis mediterranei, producing ...

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