Dual enzymatic routes to L-tyrosine and L-phenylalanine via pretyrosine in Pseudomonas aeruginosa.

Pretyrosine, an intermediate of L-tyrosine biosynthesis in blue-green algae, was found to be enzymatically formed and utilized in Pseudomonas aeruginosa. The enzymology and regulation of aromatic biosynthesis were re-evaluated in the context of these new findings, Four species of aromatic aminotranaferase were separated and partially purified. Each was reactive with prephenate, phenylpyruvate, and 4hydroxyphenylpyruvate. Molecular weights of aminotransferases L)E I and HA I were 70,000, whereas aminotransferases HA II and HA III had molecular weights of 200,000. LGlutamate was the best amino donor reactant with aminotransferase DE I, whereas I.-leucine was best with the remaining three aminotransferases. Gel filtration, DEAE-cellulose chromatography, and hydroxylapatite chromatography did not separate prephenate dehydrogenase activity from pretyrosine dehydrogenase activity (M, = 140,000). Pretyrosine dehydratase, a previously undescribed enzyme activity, converts pretyrosine to Lphenylalanine in a reaction that is analogous to the conversion of prephenate to phenylpyruvate (prephenate dehydratase). Pretyrosine dehydratase is resolved by hydroxylapatite chromatography into three subforms with similar properties and identical molecular weights (M, = 80,000). Pretyrosine dehydratase is also reactive with prephenate, functioning in uitro as prephenate dehydratase. Chorismate mutase II Ofr = 27,000) and pretyrosine dehydratase are unregulated. The bifunctional protein, chorismate mutaselprephenate dehydratase (M, = 134,000), does not appear to be an aggregate of chorismate mutase II and pretyrosine dehydratase. Both activities of the bifunctional protein are not only inhibited by L-phenylalanine, but prephenate dehydratase is activated by L-tyrosine (alteration of the apparent K,,,). Prephenatelpretyrosine dehydrogenase is feedback inhibited by L-tyrosine. Activity of 3-deoxy-oarabino-heptulosonate-7-phosphate synthetase is not influenced by pretyrosine. The dual enzymatic pathways to either L-phenylalanine or L-tyrosine provide an enzymological basis for explanation of the failure of routine mutagenesis procedures to produce auxotrophs with absolute requirements for either amino acid.