title: A reporter system that discriminates EF-hand-sensor motifs from signal-modulators at the single-motif level.
abstract: The T-protein is a single-polypeptide bi-functional enzyme composed of a chorismate mutase domain fused to a prephenate dehydrogenase domain (TyrA). We replaced the chorismate mutase domain with canonical or pseudo-Ca(2+)-binding motifs (EF-hand). Canonical-EF-hand-motifs differentiate from pseudo-EF-hand-motifs by experimenting a Ca(2+)-dependent conformational change. The Ca(2+)-free EF-hand-TyrA fusion-proteins showed TyrA activity at the T-protein level. Canonical-EF-hand-TyrA fusions showed a Ca(2+)-dependent loss of TyrA activity, but a pseudo-EF-hand-TyrA fusion showed high TyrA activity level in excess-Ca(2+) conditions. Because TyrA activity exhibits robust changes in response to Ca(2+)-dependent-EF-hand conformational alterations, TyrA could be a good Ca(2+)-reporter enzyme. A chimeric canonical/pseudo-EF-hand strategy is proposed to confer pseudo-EF-hand motifs with a Ca(2+)-dependent conformational change.
authors: Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de, Mexico. joel@ibt.unam.mx
authors: Joel
authors: J
authors: Osuna
authors: Humberto
authors: H
authors: Flores
authors: Paul
authors: P
authors: Gaytán
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011303:Prephenate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; C076398:TyrA protein, Bacteria; D011303:Prephenate Dehydrogenase; D002118:Calcium