@article{mbs:/content/journal/micro/10.1099/00221287-138-7-1309, author = "Xia, Tianhui and Zhao, Genshi and Fischer, Randy S. and Jensen, Roy A.", title = "A monofunctional prephenate dehydrogenase created by cleavage of the 5′ 109 bp of the tyrA gene from Erwinia herbicola", journal= "Microbiology", year = "1992", volume = "138", number = "7", pages = "1309-1316", doi = "https://doi.org/10.1099/00221287-138-7-1309", url = "https://www.microbiologyresearch.org/content/journal/micro/10.1099/00221287-138-7-1309", publisher = "Microbiology Society", issn = "1465-2080", type = "Journal Article", abstract = "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 tyrA. 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 tyrA 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 tyrA*, 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 α-peptide of pUC19. The TyrA* protein retained dehydrogenase activity but lacked mutase activity, thus demonstrating the separability of the two catalytic domains. While the K m of the TyrA* 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 tyrA* 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 μM to 14 μM. 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.", }