title: Probing the catalytic mechanism of prephenate dehydratase by site-directed mutagenesis of the Escherichia coli P-protein dehydratase domain.
abstract: The Escherichia coli bifunctional P-protein, which plays a central role in L-phenylalanine (Phe) biosynthesis, contains distinct chorismate mutase (CM) and prephenate dehydratase (PDT) domains as well as a regulatory (R) domain for feedback control by Phe. To elucidate the catalytic mechanism of PDT in the P-protein, 24 mutations of 15 conserved residues in the PDT domain were created, expressed in the pheA(-)E. coli strain NK6024, and studied for their effect on PDT activity. Fourteen mutant enzymes were purified to homogeneity, tested for feedback inhibition by Phe, and characterized by kinetic analysis and circular dichroism spectroscopy. Selected mutant enzymes were further studied by gel filtration, fluorescence emission, and microcalorimetry. In addition, a monofunctional PDT domain (PDT20, residues 101-285) was cloned and overexpressed in plasmid pET with expression levels up to 200-250 mg/L. PDT20 retained full PDT activity, lacked CM activity, and was insensitive to feedback inhibition by Phe. Four residues (T278, N160, Q215, and S208) were shown to be important for PDT catalysis. The values of k(cat)/K(m) for the S208A/C and T278S mutant enzymes were 100-fold lower, and 500-fold lower for the N160A and Q215A mutant enzymes than the wild-type (WT) protein. The T278A and T278V mutant enzymes displayed no measurable catalytic activity, yet bound both prephenate and a competitive inhibitor (S-DNBA) comparably to the WT protein. These data, taken together with the normal CD spectra of the mutant enzymes, strongly suggested that T278 was involved in the catalytic mechanism. To establish whether acidic residues were involved in catalysis, all the conserved Glu and Asp residues in the PDT domain were mutated to Ala. None of these mutations significantly reduced PDT activity, indicating that the acidic residues of the PDT domain are not directly involved in catalysis. However, two mutant enzymes (E159A and E232A) displayed higher levels of PDT activity (2.2- and 3.5-fold, respectively), which was due to enhanced substrate binding. For the double mutant enzyme (E159A-E232A), k(cat)/K(m) was ca. 7-fold higher than for the WT enzyme, while its K(m) was 4.6-fold lower.
authors.affiliation: Department of Molecular Biology, Baker Laboratory, Cornell University, Ithaca, New York 14853, USA.
authors.forename: S
authors.initials: S
authors.lastname: Zhang
authors.forename: D B
authors.initials: DB
authors.lastname: Wilson
authors.forename: B
authors.initials: B
authors.lastname: Ganem
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001665:Binding Sites; D002151:Calorimetry; D002384:Catalysis / Q000187:drug effects; D002826:Chorismate Mutase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002850:Chromatography, Gel; D002942:Circular Dichroism; D017124:Conserved Sequence / Q000235:genetics; D003509:Cyclohexanecarboxylic Acids / Q000378:metabolism; D053138:Cyclohexenes; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005246:Feedback / Q000187:drug effects; D005453:Fluorescence; D007700:Kinetics; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D010649:Phenylalanine / Q000378:metabolism / Q000494:pharmacology; D011302:Prephenate Dehydratase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D017186:Titrimetry
chemical_list: D001426:Bacterial Proteins; D003509:Cyclohexanecarboxylic Acids; D053138:Cyclohexenes; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; C071216:P-protein, E coli; D010649:Phenylalanine; D011302:Prephenate Dehydratase; D002826:Chorismate Mutase; C005550:prephenic acid