Complementation of a phycocyanin-bilin lyase from Synechocystis sp. PCC 6803 with a nucleomorph-encoded open reading frame from the cryptophyte Guillardia theta

Philipps-Universität Marburg, Laboratorium für Zellbiologie, Karl-von-Frisch Str,, D-35032 Marburg, Germany.
BMC Plant Biology (Impact Factor: 3.94). 02/2008; 8:56. DOI: 10.1186/1471-2229-8-56
Source: PubMed

ABSTRACT Cryptophytes are highly compartmentalized organisms, expressing a secondary minimized eukaryotic genome in the nucleomorph and its surrounding remnant cytoplasm, in addition to the cell nucleus, the mitochondrion and the plastid. Because the members of the nucleomorph-encoded proteome may contribute to essential cellular pathways, elucidating nucleomorph-encoded functions is of utmost interest. Unfortunately, cryptophytes are inaccessible for genetic transformations thus far. Therefore the functions of nucleomorph-encoded proteins must be elucidated indirectly by application of methods in genetically accessible organisms.
Orf222, one of the uncharacterized nucleomorph-specific open reading frames of the cryptophyte Guillardia theta, shows homology to slr1649 of Synechocystis sp. PCC 6803. Recently a further homolog from Synechococcus sp. PCC 7002 was characterized to encode a phycocyanin-beta155-bilin lyase. Here we show by insertion mutagenesis that the Synechocystis sp. PCC 6803 slr1649-encoded protein also acts as a bilin lyase, and additionally contributes to linker attachment and/or stability of phycobilisomes. Finally, our results indicate that the phycocyanin-beta155-bilin lyase of Synechocystis sp. PCC 6803 can be complemented in vivo by the nucleomorph-encoded open reading frame orf222.
Our data show that the loss of phycocyanin-lyase function causes pleiotropic effects in Synechocystis sp. PCC 6803 and indicate that after separating from a common ancestor protein, the phycoerythrin lyase from Guillardia theta has retained its capacity to couple a bilin group to other phycobiliproteins. This is a further, unexpected example of the universality of phycobiliprotein lyases.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Phycobiliproteins are employed by cyanobacteria, red algae, glaucophytes and cryptophytes for light-harvesting and consist of apo-proteins covalently associated with open-chain tetrapyrrole chromophores. While the majority of organisms assemble the individual phycobiliproteins into larger aggregates called phycobilisomes, members of the cryptophytes use a single phycobiliprotein which is localized in the thylakoid lumen. The cryptophyte Guillardia theta uses phycoerythrin PE545 utilizing the unusual chromophore 15,16-dihydrobiliverdin (DHBV) in addition to phycoerythrobilin (PEB). Both, the biosynthesis and the attachment of chromophores to the apo-phycobiliprotein have not yet been investigated for cryptophytes. In the present 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 for 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, PEB). Site directed mutagenesis identified residues Glu-136 and Arg-146 being involved in phycobilin binding. Based on the crystal structure, a model for the interaction of GtCPES with the apo-phycobiliprotein CpeB is proposed and discussed.
    Journal of Biological Chemistry 09/2014; DOI:10.1074/jbc.M114.591131 · 4.60 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Pigmentation of light-harvesting phycobiliproteins of cyanobacteria requires covalent attachment of open-chain tetrapyrroles, bilins, to the apoproteins. Thioether formation via addition of a cysteine residue to the 3-ethylidene substituent of bilins is mediated by lyases. T-type lyases are responsible for attachment to Cys155 of phycobiliprotein β-subunits. We present crystal structures of CpcT (All5339) from Nostoc sp. PCC7120 and its complex with phycocyanobilin, at 1.95 and 2.50 Angstrom resolution, respectively. CpcT forms a dimer and adopts a calyx-shaped β-barrel fold. While the overall structure of CpcT is largely retained upon chromophore binding, arginine residues at the opening of the binding pocket undergo major rotameric rearrangements anchoring the propionate groups of phycocyanobilin. Based on the structure and mutational analysis, a reaction mechanism is proposed that accounts for chromophore stabilization, and regio- and stereospecificity of the addition reaction. At the dimer interface, a loop extending from one subunit partially shields the opening of the phycocyanobilin binding pocket in the other subunit. Deletion of the loop or disruptions of the dimer interface significantly reduce CpcT lyase activity, suggesting functional relevance of the dimer. Dimerization is further enhanced by chromophore binding. The chromophore is largely buried in the dimer but in the monomer the 3-ethylidene group is accessible for the apo-phycobiliprotein, preferentially from the chromophore α-side. Asp163 and Tyr65 at the β- and α-face near the E-configured ethylidene group, respectively, support the acid-catalyzed nucleophilic Michael addition of cysteine-155 of the apoprotein to an N-acylimmonium intermediate proposed by K. Grubmayr and U.G. Wagner (Monatsh. Chem. 119, 965, 1988).
    Journal of Biological Chemistry 07/2014; 289(39). DOI:10.1074/jbc.M114.586743 · 4.60 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Phycocyanin is an important component of the phycobilisome, which is the principal light-harvesting complex in cyanobacteria. The covalent attachment of the phycocyanobilin chromophore to phycocyanin is catalyzed by the enzyme phycocyanin lyase. The photosynthetic properties and phycobilisome assembly state were characterized in wild type and two mutants which lack holo-α-phycocyanin. Insertional inactivation of the phycocyanin α-subunit lyase (ΔcpcF mutant) prevents the ligation of phycocyanobilin to α-phycocyanin (CpcA), while disruption of the cpcB/A/C2/C1 operon in the CK mutant prevents synthesis of both apo-α-phycocyanin (apo-CpcA) and apo-β-phycocyanin (apo-CpcB). Both mutants exhibited similar light saturation curves under white actinic light illumination conditions, indicating the phycobilisomes in the ΔcpcF mutant are not fully functional in excitation energy transfer. Under red actinic light illumination, wild type and both phycocyanin mutant strains exhibited similar light saturation characteristics. This indicates that all three strains contain functional allophycocyanin cores associated with their phycobilisomes. Analysis of the phycobilisome content of these strains indicated that, as expected, wild type exhibited normal phycobilisome assembly and the CK mutant assembled only the allophycocyanin core. However, the ΔcpcF mutant assembled phycobilisomes which, while much larger than the allophycocyanin core observed in the CK mutant, were significantly smaller than phycobilisomes observed in wild type. Interestingly, the phycobilisomes from the ΔcpcF mutant contained holo-CpcB and apo-CpcA. Additionally, we found that the large form of FNR (FNRL) accumulated to normal levels in wild type and the ΔcpcF mutant. In the CK mutant, however, significantly less FNRL accumulated. FNRL has been reported to associate with the phycocyanin rods in phycobilisomes via its N-terminal domain, which shares sequence homology with a phycocyanin linker polypeptide. We suggest that the assembly of apo-CpcA in the phycobilisomes of ΔcpcF can stabilize FNRL and modulate its function. These phycobilisomes, however, inefficiently transfer excitation energy to Photosystem II.
    PLoS ONE 08/2014; 9(8):e105952. DOI:10.1371/journal.pone.0105952 · 3.53 Impact Factor

Full-text (3 Sources)

Available from
May 21, 2014