title: Key residues for the light regulation of the blue light-activated adenylyl cyclase from Beggiatoa sp.
abstract: Photoactivated adenylyl cyclases are powerful tools for optogenetics and for investigating signal transduction mechanisms in biological photoreceptors. Because of its large increase in enzyme activity in the light, the BLUF (blue light sensor using flavin adenine dinucleotide)-activated adenylyl cyclase (bPAC) from Beggiatoa sp. is a highly attractive model system for studying BLUF domain signaling. In this report, we studied the influence of site-directed mutations within the BLUF domain on the light regulation of the cyclase domain and determined key elements for signal transduction and color tuning. Photoactivation of the cyclase domain is accomplished via strand β5 of the BLUF domain and involves the formation of helical structures in the cyclase domain as assigned by vibrational spectroscopy. In agreement with earlier studies, we observed severely impaired signaling in mutations directly on strand β5 as well as in mutations affecting the hydrogen bond network around the flavin. Moreover, we identified a bPAC mutant with red-shifted absorbance and a decreased dark activity that is highly valuable for long-term optogenetic experiments. Additionally, we discovered a mutant that forms a stable neutral flavin semiquinone radical in the BLUF domain and surprisingly exhibits an inversion of light activation.
authors.affiliation: Institut für Biologie/Experimentelle Biophysik, Humboldt Universität zu Berlin , Invalidenstrasse 42, D-10115 Berlin, Germany.
authors.forename: Manuela
authors.initials: M
authors.lastname: Stierl
authors.forename: Alfons
authors.initials: A
authors.lastname: Penzkofer
authors.forename: John T M
authors.initials: JT
authors.lastname: Kennis
authors.forename: Peter
authors.initials: P
authors.lastname: Hegemann
authors.forename: Tilo
authors.initials: T
authors.lastname: Mathes
mesh_terms: D000262:Adenylyl Cyclases / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects*; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects*; D053524:Beggiatoa / Q000201:enzymology* / Q000235:genetics / Q000528:radiation effects; D020134:Catalytic Domain; D004789:Enzyme Activation / Q000528:radiation effects; D008027:Light; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D062308:Optogenetics; D055668:Photochemical Processes; D020283:Photoreceptors, Microbial / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000528:radiation effects; D015398:Signal Transduction
chemical_list: D001426:Bacterial Proteins; D020283:Photoreceptors, Microbial; D011994:Recombinant Proteins; D000262:Adenylyl Cyclases