Crystal structure of phenylalanine-regulated 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli

Background: In microorganisms and plants the first step in the common pathw ay leading to the biosynthesis of aromatic compounds is the stereospecific condensation of phosphoenolpyruvate (PEP) and D-erythrose-4-phosphate (E4P) giving rise to 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP). This re action is catalyzed by DAMP synthase (DAHPS), a metal-activated enzyme, whi ch in microorganisms is the target for negative-feedback regulation by path way intermediates or by end products. In Escherichia coli there are three D AMPS isoforms, each specifically inhibited by one of the three aromatic ami no acids, Results: The crystal structure of the phenylalanine-regulated form of DAMPS complexed with PEP and Pb2+ (DAHPS(Phe)-PEP-Pb) was determined by multiple wavelength anomalous dispersion phasing utilizing the anomalous scattering of Pb2+, The tetramer consists of two tight dimers. The monomers of the ti ght dimer are coupled by extensive interactions including a pair of three-s tranded, intersubunit beta sheets. The monomer (350 residues) is a (beta/al pha)(8) barrel with several additional beta strands and a helices. The PEP and Pb2+ are at the C-ends of the beta strands of the barrel, as is SO42-, inferred to occupy the position of the phosphate of E4P. Mutations that red uce feedback inhibition cluster about a cavity near the twofold axis of the tight dimer and are centered, similar to 15 Angstrom from the active site, indicating the location of a separate regulatory site. Conclusions: The crystal structure of DAHPS(Phe)-PEP-Pb reveals the active site of this key enzyme of aromatic biosynthesis and indicates the probable site of inhibitor binding. This is the first reported structure of a DAMPS ; the structure of its two paralogs and of a variety of orthologs should no w be readily determined by molecular replacement.