Abstract
Phycobiliproteins are employed by cyanobacteria, red algae, glaucophytes, and cryptophytes for light-harvesting and consist of apoproteins covalently associated with open-chain tetrapyrrole chromophores. Although the majority of organisms assemble the individual phycobiliproteins into larger aggregates called phycobilisomes, members of the cryptophytes use a single type of phycobiliprotein that is localized in the thylakoid lumen. The cryptophyte Guillardia theta (Gt) uses phycoerythrin PE545 utilizing the uncommon chromophore 15,16-dihydrobiliverdin (DHBV) in addition to phycoerythrobilin (PEB). Both the biosynthesis and the attachment of chromophores to the apophycobiliprotein have not yet been investigated for cryptophytes. In this study, we identified and characterized enzymes involved in PEB biosynthesis. In addition, we present the first in-depth biochemical characterization of a eukaryotic phycobiliprotein lyase (GtCPES). Plastid-encoded HO (GtHo) was shown to convert heme into biliverdin IXα providing the substrate with a putative nucleus-encoded DHBV:ferredoxin oxidoreductase (GtPEBA). A PEB:ferredoxin oxidoreductase (GtPEBB) was found to convert DHBV to PEB, which is the substrate for the phycobiliprotein lyase GtCPES. The x-ray structure of GtCPES was solved at 2.0 Å revealing a 10-stranded β-barrel with a modified lipocalin fold. GtCPES is an S-type lyase specific for binding of phycobilins with reduced C15=C16 double bonds (DHBV and PEB). Site-directed mutagenesis identified residues Glu-136 and Arg-146 involved in phycobilin binding. Based on the crystal structure, a model for the interaction of GtCPES with the apophycobiliprotein CpeB is proposed and discussed.
Highlights
Cryptophytes like Guillardia theta utilize soluble phycobiliproteins for light-harvesting
Phycobilin Biosynthesis in the Cryptophyte G. theta—To obtain insights into phycobilin biosynthesis in the cryptophyte G. theta, the potentially involved plastid encoded heme oxygenase GtHo,4 and the nucleus-encoded ferredoxin-dependent bilin reductases (FDBR) GtPEBA and GtPEBB were heterologously produced in E. coli and subsequently examined with regard to their enzymatic activity
Expression of StrepII-tagged GtHo in E. coli in LB medium supplemented with the osmolytes betaine and sorbitol led to green-colored cells, indicating that StrepIIGtHo was able to convert heme produced by E. coli yielding the green-colored open-chain tetrapyrrole BV IX␣ in vivo
Summary
Cryptophytes like Guillardia theta utilize soluble phycobiliproteins for light-harvesting. Results: Guillardia theta adopted phycoerythrobilin biosynthesis from cyanobacteria, and the phycobiliprotein lyase GtCPES provides structural requirements for transfer of this chromophore to a specific cysteine residue of the apophycobiliprotein. The cryptophyte Guillardia theta (Gt) uses phycoerythrin PE545 utilizing the uncommon chromophore 15,16-dihydrobiliverdin (DHBV) in addition to phycoerythrobilin (PEB). Both the biosynthesis and the attachment of chromophores to the apophycobiliprotein have not yet been investigated for cryptophytes. Cryptophytes, cyanobacteria, red algae, and glaucophytes perform oxygenic photosynthesis using chlorophyll-containing antenna complexes and additional light-harvesting proteins, termed phycobiliproteins (PBP)3 [1, 2]. We present the first crystal structure of a eukaryotic PBP lyase, the CpeS lyase from G. theta (GtCPES) This lyase was characterized in terms of phycobilin specificity, affinity, and binding kinetics. GtCPES belongs to the clade of S/U-type lyases and is restricted to binding of DHBV and PEB
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