Regulation of prephenate dehydratase in coryneform species of bacteria by l-phenylalanine and by remote effectors

Abstract

Corynebacterium glutamicum, Brevibacterium flavum, and B. ammoniagenes utilize the phenylpyruvate branchlet as the sole means of l-phenylalanine biosynthesis. Enzymological aspects of l-phenylalanine biosynthesis were generally similar in the three microbial species selected to represent the coryneform grouping. Thus, the general picture of aromatic biosynthesis in coryneform bacteria is very similar to that found in species of bluegreen algae, i.e., phenylpyruvate branchlet for l-phenylalanine biosynthesis and pretyrosine branchlet for l-tyrosine biosynthesis ( A. Fazel and R. A. Jensen, 1979, J. Bacteriol. 138, 805–815). Prephenate dehydratase is subject to repression control and to feedback inhibition by l-phenylalanine ( K i value range: 35–95 μ m). Extrapathway effectors activated ( l-tyrosine) or inhibited ( l-tryptophan) prephenate dehydratase, reminiscent of the action of remote effectors with Bacillus subtilis prephenate dehydratase. A labile activation of prephenate dehydratase by l-methionine was also found in C. glutamicum. Prephenate dehydratase copurifies with an aromatic aminotransferase which is capable of transaminating phenylpyruvate, 4-hydroxyphenylpyruvate, or prephenate in vitro. The possibility of a multifunctional protein was ruled out, however, by results obtained with l-phenylalanine auxotrophs lacking prephenate dehydratase and a regulatory mutant constitutive for prephenate dehydratase. Other classes of regulatory mutants altered in prephenate dehydratase lost sensitivity to inhibition by l-phenylalanine (and l-tryptophan), or produced an enzyme that was hypersensitive to l-tyrosine activation. Molecular weight estimates of the prephenate dehydratase enzymes isolated from the three species studied ranged from 200,000–260,000. Unlike the B. subtilis enzyme, neither allosteric (positive or negative) effectors nor regulatory mutations altered the molecular weight of prephenate dehydratase. However, the intrinsic activity of prephenate dehydratase and its sensitivity to allosteric effectors was altered substantially following the initial step of partial purification.