Enzymological Basis for Growth Inhibition by l -Phenylalanine in the Cyanobacterium Synechocystis sp. 29108

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Abstract
The pattern of allosteric control in the biosynthetic pathway for aromatic amino acids provides a basis to explain vulnerability to growth inhibition by L-phenylalanine (0.2 mM or greater) in the unicellular cyanobacterium Synechocystis sp. 29108. Growth inhibition was attributed to the hypersensitivity of 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase to feedback inhibition by L-phenylalanine. Hyperregulation of this initial enzyme of aromatic biosynthesis depletes the supply of precursors needed for biosynthesis of L-tyrosine and L-tryptophan. Consistent with this mechanism is the total reversal of phenylalanine inhibition by a combination of tyrosine and tryptophan. Inhibited cultures also contained decreased levels of phycocyanin pigments, a characteristic previously correlated with amino acid starvation in cyanobacteria. L-Phenylalanine is a potent noncompetitive inhibitor (with both substrates) of 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase, whereas L-tyrosine is a very weak inhibitor. Prephenate dehydratase also displays allosteric sensitivity to phenylalanine (inhibition) and to tyrosine (activation). Both 2-fluoro and 4-fluoro derivatives of phenylalanine were potent analog antimetabolites, and these were used in addition to L-phenylalanine as selective agents for resistent mutants. Mutants were isolated which excreted both phenylalanine and tyrosine, the consequence of an altered 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase no longer sensitive to feedback inhibition. Simultaneous insensitivity to L-tyrosine suggests that L-tyrosine acts as a weak analog mimic of L-phenylalanine at a common binding site. Prephenate dehydratase in the regulatory mutants was unaltered. Such mutants excrete more phenylalanine than tyrosine, indicating that L-tyrosine activation dominates L-phenylalanine inhibition of prephenate dehydratase in vivo. In mutant Phe r19 the loss in allosteric sensitivity of 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase was accompanied by a 3-fold increase in specific activity. This could suggest that existence of a modest degree of repression control (autogenous) over 3-deoxy-D-arabinoheptulosonate synthase, although other explanations are possible.