A MONOFUNCTIONAL PREPHENATE DEHYDROGENASE CREATED BY CLEAVAGE OF THE 5' 109 BP OF THE TYRA GENE FROM ERWINIA-HERBICOLA

A cohesive phylogenetic cluster that is limited to enteric bacteria and a few closely related genera possesses a bifunctional protein that is known as the T-protein and is encoded by tyr A. The T-protein carries catalytic domains for chorismate mutase and for cyclohexadienyl dehydrogenase. Cyclohexadienyl dehydrogenase can utilize prephenate or L-arogenate as alternative substrates. A portion of the tyr A gene cloned from Erwinia herbicola was deleted in vitro with exonuclease III and fused in-frame with a 5' portion of lacZ to yield a new gene, denoted tyr A*, in which 37 N-terminal amino acids of the T-protein are replaced by 18 amino acids encoded by the polycloning site/5' portion of the lacZ alpha-peptide of pUC19. The Tyr A* protein retained dehydrogenase activity but lacked mutase activity, thus demonstrating the separability of the two catalytic domains. While the K(m) of the Tyr A* dehydrogenase for NAD+ remained unaltered, the K(m) for prephenate was fourfold greater and the V(max) was almost twofold greater than observed for the parental T-protein dehydrogenase. Activity with L-arogenate, normally a relatively poor substrate, was reduced to a negligible level. The prephenate dehydrogenase activity encoded by tyr A* was hypersensitive to feedback inhibition by L-tyrosine (a competitive inhibitor with respect to prephenate), partly because the affinity for prephenate was reduced and partly because the K(i) value for L-tyrosine was decreased from 66-mu-M to 14-mu-M. Thus, excision of a portion of the chorismate mutase domain is shown to result in multiple extra-domain effects upon the cyclohexadienyl dehydrogenase domain of the bifunctional protein. These include alterations in apparent substrate specificity, isoelectric point, stability, catalytic properties and regulatory properties.