Mapping of chorismate mutase and prephenate dehydrogenase domains in the Escherichia coli T-protein.
The Escherichia coli bifunctional T-protein transforms chorismic acid to p-hydroxyphenylpyruvic acid in the l-tyrosine biosynthetic pathway. The 373 amino acid T-protein is a homodimer that exhibits chorismate mutase (CM) and prephenate dehydrogenase (PDH) activities, both of which are feedback-inhibited by tyrosine. Fifteen genes coding for the T-protein and various fragments thereof were constructed and successfully expressed in order to characterize the CM, PDH and regulatory domains. Residues 1-88 constituted a functional CM domain, which was also dimeric. Both the PDH and the feedback-inhibition activities were localized in residues 94-373, but could not be separated into discrete domains. The activities of cloned CM and PDH domains were comparatively low, suggesting some cooperative interactions in the native state. Activity data further indicate that the PDH domain, in which NAD, prephenate and tyrosine binding sites were present, was more unstable than the CM domain.
Chen
Shuqing
S
Department of Molecular Biology and Genetics, Cornell University, NY 14853, USA.
Vincent
Sarah
S
Wilson
David B
DB
Ganem
Bruce
B
D001665:Binding Sites; D002826:Chorismate Mutase / Q000235:genetics / Q000378:metabolism*; D002827:Chorismic Acid / Q000378:metabolism; D003001:Cloning, Molecular; D003509:Cyclohexanecarboxylic Acids / Q000378:metabolism; D053138:Cyclohexenes; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D025461:Feedback, Physiological; D015964:Gene Expression Regulation, Bacterial; D010667:Phenylpyruvic Acids / Q000378:metabolism; D011303:Prephenate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014443:Tyrosine / Q000494:pharmacology*
D003509:Cyclohexanecarboxylic Acids; D053138:Cyclohexenes; D029968:Escherichia coli Proteins; D010667:Phenylpyruvic Acids; D011994:Recombinant Proteins; C010590:4-hydroxyphenylpyruvic acid; D014443:Tyrosine; D011303:Prephenate Dehydrogenase; D002826:Chorismate Mutase; D002827:Chorismic Acid; C005550:prephenic acid