title: Indoleglycerol phosphate synthase-phosphoribosyl anthranilate isomerase: comparison of the bifunctional enzyme from Escherichia coli with engineered monofunctional domains.
abstract: Putative domain--domain interactions of the monomeric bifunctional enzyme indoleglycerol phosphate synthase:phosphoribosyl anthranilate isomerase from Escherichia coli were probed by separating the domains on the gene level and expressing them as monofunctional proteins. The engineered monofunctional enzymes were found to be stable, monomeric proteins with virtually full catalytic activity. In addition, binding of indolyglycerol phosphate to the active site of indoleglycerol phosphate synthase and binding of reduced 1-[(2-carboxyphenyl)amino]-1-deoxyribulose 5-phosphate, a competitive inhibitor of both indoleglycerol phosphate synthase and phosphoribosyl anthranilate isomerase, were almost identical in both the mono- and bifunctional enzymes. Furthermore, no association between the monofunctional enzymes was found, neither in vitro, by sedimentation and gel filtration experiments, nor in vivo, by coexpression of the domains in the same cell. Thus, no selective advantages of the bifunctional enzyme from Escherichia coli over the respective monofunctional enzymes were found on a functional level. However, the phosphoribosyl anthranilate isomerase domain appears to stabilize the indoleglycerol phosphate synthase domain of the bifunctional enzyme from Escherichia coli by interactions that seem to subtly influence the kinetics of ligand binding.
authors.affiliation: Abteilung Biophysikalische Chemie, Biozentrum der Universität Basel, Switzerland.
authors.forename: M
authors.initials: M
authors.lastname: Eberhard
authors.forename: M
authors.initials: M
authors.lastname: Tsai-Pflugfelder
authors.forename: K
authors.initials: K
authors.lastname: Bolewska
authors.forename: U
authors.initials: U
authors.lastname: Hommel
authors.forename: K
authors.initials: K
authors.lastname: Kirschner
mesh_terms: D019747:Aldose-Ketose Isomerases; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002238:Carbohydrate Epimerases / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D005798:Genes, Bacterial; D007209:Indole-3-Glycerol-Phosphate Synthase / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D009876:Operon; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity
chemical_list: D009097:Multienzyme Complexes; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; C052909:phosphoribosylanthranilate isomerase-indoleglycerol-phosphate synthase; D007209:Indole-3-Glycerol-Phosphate Synthase; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases