Probing the catalytic mechanism of prephenate dehydratase by site-directedmutagenesis of the Escherichia coli P-protein dehydratase domain

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) doma in 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 fo r their effect on PDT activity. Fourteen mutant enzymes were purified to ho mogeneity, tested for feedback inhibition by Phe, and characterized by kine tic analysis and circular dichroism spectroscopy. Selected mutant enzymes w ere further studied by gel filtration, fluorescence emission, and microcalo rimetry. In addition, a monofunctional PDT domain (PDT20, residues 101-285) was cloned and overexpressed in plasmid pET with expression levers up to 2 00-250 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(ca t)/K-m for the S208A/C and T278S mutant enzymes were 100-fold lower, and 50 0-fold lower for the N160B and Q215A mutant enzymes than the wild-type (WT) protein. The T278A and T278V mutant enzymes displayed no measurable cataly tic 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 invo lved in catalysis, all the conserved Glu and Asp residues in the PDT domain were mutated to Ala. None of these mutations significantly reduced PDT act ivity, indicating that the acidic residues of the PDT domain are not direct ly involved in catalysis. However, two mutant enzymes (E159A and E232A) dis played higher levels of PDT activity (2.2- and 3.5-fold, respectively), whi ch was due to enhanced substrate binding. For the double mutant enzyme (E15 9A-E232A), k(cat)/K-m was ca. 7-fold higher than for the WT enzyme, while i ts K-m was 4.G-fold lower.