Probing the potential metal binding site in Escherichia coli 3-deoxy- d- arabino-heptulosonate 7-phosphate synthase (phenylalanine-sensitive)

• Published in: FEBS letters Vol. 441; no. 2; pp. 195 - 199
• Main Authors: Sundaram, Appavu K, Howe, David L, Sheflyan, Galina Ya, Woodard, Ronald W
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 18.12.1998
• Subjects: 3-Deoxy-7-Phosphoheptulonate Synthase - chemistry; 3-Deoxy-7-Phosphoheptulonate Synthase - genetics; 3-Deoxy-7-Phosphoheptulonate Synthase - metabolism; Base Sequence; Binding Sites; BTP, 1,3-bis[tris-(hydroxymethyl)-methylamino]propane; DAH 7-P synthase, 3-deoxy-d-arabino-heptulosonic acid 7-phosphate synthase; DAH 7-P, 3-deoxy-d-arabino-heptulosonic acid 7-phosphate; Deoxy- d- arabino-heptulosonate 7-phosphate synthase; DNA Primers; E 4-P, erythrose 4-phosphate; EDTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; Erythrose 4-phosphate; Escherichia coli - enzymology; Kinetics; Metal binding; Metals - metabolism; Molecular Probes; Mutagenesis, Site-Directed; PEP, phosphoenolpyruvate; Phenylalanine - pharmacology; Phospho enolpyruvate; Phosphoenolpyruvate; Site-directed mutagenesis; Erythrose 4-phosphate; Phospho enolpyruvate; EDTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; Site-directed mutagenesis; DAH 7-P synthase, 3-deoxy- d- arabino-heptulosonic acid 7-phosphate synthase; PEP, phospho enolpyruvate; Metal binding; Deoxy- d- arabino-heptulosonate 7-phosphate synthase; E 4-P, erythrose 4-phosphate; DAH 7-P, 3-deoxy- d- arabino-heptulosonic acid 7-phosphate; BTP, 1,3-bis[tris-(hydroxymethyl)-methylamino]propane
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/S0014-5793(98)01545-2

The active site residues of the proposed metal binding site of DAH 7-P synthase (phe) were probed by site-directed mutagenesis of C61 to glycine and serine, H64 to glycine, and with the double mutant C61H/H64C. While C61S and C61H/H64C were inactive, both C61G and H64G were active. All mutants, regardless of enzymatic activity, bound one equivalent of Fe 2+ per monomeric unit. Even though C61 and H64 were shown not to be metal ligands for the DAH 7-P synthase (phe), they may provide some of the backbone interactions/secondary structural elements necessary to properly form the metal binding pocket.