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 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 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 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 alpha-peptide of pUC19. The TyrA* protein retained dehydrogenase activity but lacked mutase activity, thus demonstrating the separability of the two catalytic domains. While the Km of the TyrA* dehydrogenase for NAD+ remained unaltered, the Km for prephenate was fourfold greater and the Vmax 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 Ki value for L-tyrosine was decreased from 66 microM to 14 microM. 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Xia
T
T
Department of Microbiology and Cell Science, University of Florida, Gainesville 32611.
Zhao
G
G
Fischer
R S
RS
Jensen
R A
RA
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004885:Erwinia / Q000201:enzymology / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D011303:Prephenate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D009097:Multienzyme Complexes; C076398:TyrA protein, Bacteria; D011303:Prephenate Dehydrogenase