Phycobiliprotein Lyases

Tu, Jun-Ming (2008): Phycobiliprotein Lyases: Structure of Reconstitution Products and Mechanistic Studies. Dissertation, LMU München: Fakultät für Biologie

Abstract

Phycobilins are light harvesting pigments of cyanobacteria and red algae. In cyanobacteria, four phycobiliproteins are organized in phycobilisomes: phycocyanin (PC), allophycocyanin (APC), and often also phycoerythrocyanin (PEC) or phycoerythrin (PE). Their phycobilin chromophores, linear tetrapyrroles, are generally bound to the apoprotein at conserved positions by cysteinyl thioether linkages. A final step in phycobiliprotein biosynthesis is the post-translational phycobilin addition to the various biliproteins. In vivo, the correct attachment of most chromophores is catalyzed by binding-site and chromophore-specific lyases. Only two such lyases, which both belong to the E/F-type were known at the beginning of this work. Two additional types, S/(U)-type and T-type lyase, have been characterized during this work. In addition, the correct structures of the products from all three lyase types have been verified, and evidence was obtained for the reaction mechanisms. This characterization relied on two methodological advances. The first is the use of a multi-plasmidic expression system for reconstitution of phycobiliproteins in E. coli. After cloning of apophycobiliprotein genes, phycobilin biosynthesis genes and (putative) lyase genes from several cyanobacteria, various phycobiliproteins could be biosynthesized in the heterologous E. coli system using dual plasmids containing the respective genes. This heterologous system produces higher yields than the in vitro reconstitution, it is nearly devoid of spontaneous binding, better reproducible, and more easily controlled. The second methodological advance is the consequent use of a combination of chromatographic, electrophoretic and spectroscopic tools that allowed a full characterization of the structure and binding sites of attached chromophores. This included, besides optical spectroscopy, in particular mass and magnetic resonance (1H-NMR) spectroscopy. Using the unmodified genes coding for both subunits of PEC, as well as their cystein mutants, three lyases were identified for the three binding site. Besides the already known isomerizing lyase, PecE/PecF, for Cys-84 of α-PEC, these are the two new lyases, CpcT (all5339) for Cys-153 of β-PEC, and CpcS (alr0617) for Cys-82 of β-PEC. The spectroscopic analysis proved that the chromophores (PCB and PVB)are correctly attached to these three binding sites. Similarly, three lyases were identified for the three binding sites of CPC. The well known heterodimeric lyase (CpcE/CpcF) catalyzes the covalent attachment of PCB to αC84 of CPC, CpcS catalyses the site-selective attachment of PCB to cysteine-β84 in CpcB; and CpcT for cysteine-β155 of CpcB. CpcE/F is specific for CpcA, while CpcS and CpcT can react with both CpcB and PecB. We also tested the lyase activity of the deoxyhyposyl-hydroxylase (DOHH) from the malaria parasite, Plasmodium falciparum. This enzyme has Heat-like repeats that are characteristic for the E/F-type lyases, but it had not chromophore-attaching activity. The substrate specificity of the new lyase, CpcS (coded by alr0617), was further tested with APC subunits; It is very unspecific with regard to the acceptor protein and attaches PCB to ApcA1, ApcB, ApcD ApcF, as well as to the product of an additional gene, apcA2; of unknown function that is highly homologous to apcA1 coding for the APC α-subunit. Obviously, this lyase has a much broader substrate specificity than the E/F-type lyases, but it has high site-specificity, attaching the chromophore exclusively to the Cys-84 (consensus sequence) binding site of the APC subunits. CpcS from Anabaena PCC7120 is a relatively simple system, it acts as a monomer, and does not require any cofactors. CpcS binds PCB rapidly (<1s) and relatively strongly, but probably non-covalently. The chromophore is bound in an extended conformation similar to that in phycobiliproteins, but only poorly fluorescent. The extended conformation is supported by binding studies with a conformationally locked chromophore, 15Za-PCB, which also binds rapidly and non-covalently to CpcS and gives a product with similar spectral properties as PCB-CpcS. Upon addition of apo-biliproteins to the PCB-CpcS (or 15Za-PCB-CpcS) complex, the chromophore is transferred to the latter much more slowly (~1 hr), indicating that chromophorylated CpcS is an intermediate in the enzymatic reaction. There are distinct differences in the absorption, extinction coefficient in acidic methanol and pKa of the free 15Za-PCB, compared with that of free PCB, which are probably due to a shift in the pK-values by about 1 pH unit. Nucleophilic addition products of PCB were characterized that are formed spontaneously or by the lyases, and gave first indications for a mechanistic model for the lyases. The first nucleophile was imidazole, which is a model for histidine. Two imidazole-PCB adducts were prepared and the structures determined by MS and NMR spectroscopy. Surprisingly, the chromophore is isomerized in this reaction to a 2,22 H –bilin termed iso-phycocyanobilin (iPCB). CpcS not only can promote covalent binding of PCB to imidazole, but also catalyses the transfer of the chromophore of the formed iPCB-imidazole to the cysteine84 of acceptor apoprotein, CpcB. During this transfer reaction, the chromophore is re-isomerized to PCB, to yield CpcB-C84-PCB. It indicates that chromophorylation by CpcS might then involve a histidine-bound intermediate; this could be a model for the reaction catalyzed by CpcS. The second nucleophile was mercaptoethanol, as a model for cysteine. In the ME and PCB reaction system, two isomers each of isomeric PVB-ME and iPCB-ME were obtained in a non-enzymatic reaction. The chromophore of the two complexes can be transferred to cysteine-84 of CpcB, yielding CpcB-C84-PCB and CpcB-C84-PVB. In the presence of the lyase, CpcS, only the iPCB adducts are formed. It indicates autocatalytical chromophorylation might then involve a thiol-chromophore intermediate; this could be a model for the chromophorylation reaction. At the same, we propose a possible generalized catalytic mechanism for the non-isomerizing heterodimeric lyase, CpcE/CpcF, and its isomerizing homolog, PecE/PecF.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: phycobiliprotein lyase, reconstitution, mechanism
• Themengebiete: 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie ; 500 Naturwissenschaften und Mathematik
• Fakultäten: Fakultät für Biologie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 9. September 2008
• 1. Bericht­erstatter:in: Scheer, Hugo