Biosynthesis of phycocyanobilin in photosynthetic eukaryotic algae

• Applicants: Professorin Dr. Nicole Frankenberg-Dinkel; Professor Dr. Eckhard Hofmann
• Subject Area: Biochemistry ; Plant Biochemistry and Biophysics
• Term: from 2014 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 256368057

Open-chain tetrapyrrole pigments (bilins) are widely distributed in many organisms where they serve distinct functions. Cyanobacteria, red algae and cryptophytes are organisms with the highest concentration of (phyco)bilins. In these organisms, bilins are employed for both, light-harvesting (phycobiliproteins) and light-sensing (phytochromes). Within this grant proposal we will investigate unusual biosynthetic pathways to the turquoise phycobilin phycocyanobilin (PCB). While cyanobacteria use a single enzyme of the ferredoxin-dependent bilin reductases (FDBR) to convert biliverdin IXalpha in a four-electron reducing step to PCB, some algae lack this enzyme although they contain significant amounts of this pigments in either their phycobiliproteins or in the light sensing phytochromes. The red alga Galdieria sulphuraria, member of the cyanidiales, for instance only uses PCB in its light harvesting phycobiliproteins but lacks a canonical biosynthetic enzyme for PCB. However, it possesses genes encoding two FDBRs involved in the biosynthesis of the PCB-isomer phycoerythrobilin (PEB). Preliminary data suggest that PEB is indeed a biosynthetic precursor to PCB in this organism and that a thus far unknown isomerase catalyses the conversion. One major objective of this proposal is therefore to purify this enzymatic activity from G. sulphuraria by means of classical protein chromatography and with the addition of a bioinformatics approach nail down putative candidate proteins. Once identified, recombinant candidate proteins will be purified followed by structural and catalytic analysis. In a second objective, we will investigate a homolog of the plant FDBR HY2 from the streptophyte alga Klebsormidium flaccidum (KflaHY2). While the higher plant enzyme converts biliverdin IXalpha in a two-electron reduction to phytochromobilin, the algal protein is synthesizing PCB. Based on our recently solved crystal structure the FDBR PEBB, a structural model for KflaHY2 was generated suggesting that an aspartate/aspargine switch occurred within the HY2 family. Here, we wish to confirm that this amino acid switch is the reason for changed catalytic specificity by analyzing several members of this outgroup and will identify other molecular determinants for this surprising change of function. As the three-dimensional arrangement of the enzymes active site is essential for the interpretation of these functional data, we will focus our efforts towards resolving a crystal structure of KflaHY2.

• DFG Programme: Research Grants