Posttranslationale Modifikation der Phycobiliproteine aus der Cryptophyte Guillardia theta
Stoffwechselphysiologie, Biochemie und Genetik der Mikroorganismen
Zusammenfassung der Projektergebnisse
In addition to chlorophylls, cryptophytes employ phycobiliproteins (PBP) for light harvesting during oxygenic photosynthesis. PBPs are also ubiquitously distributed in cyanobacteria, red algae and glaucophytes. For light harvesting, PBP subunits carry covalently attached openchain tetrapyrrole chromophores called phycobilins. Phycobilins absorb green light and make it available for photosynthesis. Cryptophytes gained the ability for photosynthesis during secondary endosymbiosis by uptake of a former red algae. Eventually, evolutionary development resulted in modified PBPs. In contrast to the majority of organisms in which the individual PBPs are organized in larger aggregates, known as phycobilisomes, cryptophytes employ a single type of PBP, which is localized in the thylakoid lumen of the chloroplast. Guillardia theta utilizes the PBP phycoerythrin PE545 which binds one molecule 15,16-dihydrobiliverdin (DHBV) at both α subunits and three molecules of phycoerythrobilin (PEB) at β subunits. Thus far, the attachment of bilins to cryptophycean apo-PBPs is not yet completely understood. However, it is known from cyanobacteria that chromophorylation is often mediated by phycobiliprotein lyases (PBP lyases) which support phycobilin transfer. Within this project, the functional characterization of eukaryotic S-type PBP lyase GtCPES from G. theta was performed. Fluorescence spectroscopy and zinc-induced fluorescence showed that GtCPES mediates transfer and covalent attachment of 3(Z)-PEB to the conserved Cys82 of PBP-β subunit of Prochlorococcus marinus MED4 (PmCpeB). With aid of site-directed mutagenesis amino acid residues involved in phycobilin binding and transfer were identified. spectroscopic binding and transfer studies employing protein variants identified three amino acid residues within the binding pocket that are relevant for PEB binding (Trp69, Glu136, Glu168). All three residues are likely involved in PEB coordination in the binding pocket to stabilize its conformation. In addition, two amino acid residues involved in PEB transfer were identified (Trp75, Ser150). Here, Trp75 is essential for the transfer reaction. Moreover, one single amino acid residue (Met67) was identified whose exchange resulted in a larger binding pocket that allowed 3(E)-phycocyanobilin (PCB) to bind. Thus, Met67 is responsible for the restricted substrate specificity of GtCPES, which is usually limited to PEB and its biosynthetic precursor 15, 16-dihydrobiliverdin. GtCPES_M67A binds both PEB and PCB in a stable, colorful complex in vitro and in vivo when produced in Escherichia coli. Furthermore, GtCPES_M67A is able to mediate PCB transfer to suitable apo-PBP subunits. Based on our data we postulate that a single amino acid residue determines the bilin-specificity of phycoerythrin S-type lyases but that additional factors regulate handover to the target protein.
Projektbezogene Publikationen (Auswahl)
- (2017) Dinstinct features of cyanophage-encoded T-type phycobiliprotein lyase CpeT-The role of auxiliary metabolic genes. J. Biol. Chem. 292, 3089-3098
Gasper, R., Schwach, J., Hartmann, J., Holtkamp, A., Riedel, N., Wiethaus, J., Hofmann, E. & Frankenberg-Dinkel, N.
(Siehe online unter https://doi.org/10.1074/jbc.M116.769703) - (2019) Untersuchungen zur Struktur und Spezifität der Phycobiliproteinlyase CPES aus Guillardia theta. Dissertation, Fachbereich Biologie, TU Kaiserslautern
Tomazic, N.
- (2020) Exchange of a single amino acid residue in the cryptophyte phycobiliprotein lyase GtCPES expands its substrate specificity
Tomazic, N., Overkamp, K., Aras, M., Pierik, A.J., Hofmann, E. & Frankenberg-Dinkel, N.
(Siehe online unter https://doi.org/10.1101/2020.03.31.018853)
