title: On the role of helix 0 of the tryptophan synthetase alpha chain of Escherichia coli.
abstract: The role of helix 0 of the alpha chain (TrpA) of the tryptophan synthetase alpha2beta2 multi-functional enzyme complex of Escherichia coli was examined by deleting amino-terminal residues 2-6, 2-11, or 2-19 of TrpA. Selected substitutions were also introduced at TrpA positions 2-6. The altered genes encoding these polypeptides were overexpressed from a foreign promoter on a multicopy plasmid and following insertion at their normal chromosomal location. Each deletion polypeptide was functional in vivo. However all appeared to be somewhat more labile and insoluble and less active enzymatically than wild type TrpA. The deletion polypeptides were overproduced and solubilized from cell debris by denaturation and refolding. Several were partially purified and assayed in various reactions in the presence of tryptophan synthetase beta2 (TrpB). The purified TrpADelta2-6 and TrpADelta2-11 deletion polypeptides had low activity in both the indole + serine --> tryptophan reaction and the indoleglycerol phosphate + serine --> tryptophan reaction. Poor activity in each reaction was partly due to reduced association of TrpA with TrpB. The addition of the TrpA ligands, alpha-glycerophosphate or indoleglycerol phosphate, during catalysis of the indole + serine --> tryptophan reaction increased association and activity. These findings suggest that removal of helix 0 of TrpA decreases TrpA-TrpB association as well as the activity of the TrpA active site. Alignment of the TrpA sequences from different species indicates that several lack part or all of helix 0. In some of these polypeptides, extra residues at the carboxyl end may substitute for helix 0.
authors.affiliation: Department of Biological Sciences, Stanford University, Stanford, California, 94305-5020, USA.
authors.forename: M C
authors.initials: MC
authors.lastname: Yee
authors.forename: V
authors.initials: V
authors.lastname: Horn
authors.forename: C
authors.initials: C
authors.lastname: Yanofsky
mesh_terms: D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology; D003196:Computer Graphics; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008715:Methionine / Q000378:metabolism; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D012984:Software; D014367:Tryptophan Synthase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D046911:Macromolecular Substances; D011994:Recombinant Proteins; D008715:Methionine; D014367:Tryptophan Synthase