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

Structure. 1999 Jul 15;7(7):865-75. doi: 10.1016/s0969-2126(99)80109-9.

Abstract

Background: In microorganisms and plants the first step in the common pathway 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 reaction is catalyzed by DAHP synthase (DAHPS), a metal-activated enzyme, which in microorganisms is the target for negative-feedback regulation by pathway intermediates or by end products. In Escherichia coli there are three DAHPS isoforms, each specifically inhibited by one of the three aromatic amino acids.

Results: The crystal structure of the phenylalanine-regulated form of DAHPS 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 tight dimer are coupled by extensive interactions including a pair of three-stranded, intersubunit beta sheets. The monomer (350 residues) is a (beta/alpha)8 barrel with several additional beta strands and alpha helices. The PEP and Pb2+ are at the C-ends of the beta strands of the barrel, as is SO4(2-), inferred to occupy the position of the phosphate of E4P. Mutations that reduce feedback inhibition cluster about a cavity near the twofold axis of the tight dimer and are centered approximately 15 A 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 DAHPS; the structure of its two paralogs and of a variety of orthologs should now be readily determined by molecular replacement.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 3-Deoxy-7-Phosphoheptulonate Synthase / antagonists & inhibitors
  • 3-Deoxy-7-Phosphoheptulonate Synthase / chemistry*
  • 3-Deoxy-7-Phosphoheptulonate Synthase / metabolism
  • Amino Acid Sequence
  • Binding Sites
  • Catalysis
  • Crystallography, X-Ray
  • Escherichia coli / enzymology*
  • Models, Molecular
  • Molecular Sequence Data
  • Phenylalanine / metabolism*
  • Protein Conformation
  • Sequence Homology, Amino Acid

Substances

  • Phenylalanine
  • 3-Deoxy-7-Phosphoheptulonate Synthase

Associated data

  • PDB/1QR7