Phycobiliprotein Lyases

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 (