Studies On The Character And Catalytic Function Of Phycobiliprotein Lyases From Mastigocladus Laminosus

Phycobiliproteins are a homologous family of light-harvesting proteins present in cyanobacteria, red algae, and cryptophytes. The last step in phycobilin biosynthesis is the phycobilin addition to the apophycobiliproteins. In vivo, the correct attachment of most chromophores is catalyzed by lyases, of which only few have hitherto been characterized. As we know now, PecE and PecF catalyzed both the attachment and isomerization of phycocyanobilin to PecA, while the covalent attachment of phycocyanobilin to the α-subunit of C-phycocyanin, CpcA, is catalyzed by CpcE and CpcF. PecE/F and CpcE/F showed 20⁴0% homologous. Neither the mechanism is known of the ligation catalyzed by the CpcE/F, nor of the ligation/isomerization catalyzed by PecE/F. In this work, we used the ways of deletion mutation, site-directed mutations and chemical modification. PecE/F and CpcE/F were studied in nearly the same ways to compare their mechanisms on catalyzing the same substrate PCB. PecE/F from M. laminosus, was first studied on its cofactor in the reconstitution system in vitro. Mercaptoethanol(5mmol/L) and the divalent metals, Mg²⁺(5mmol/L) or Mn²⁺(3mmol/L), were required, and the reaction was aided by 1% Triton X-100. Phosphate and Tris?HCl mixed buffer was better for the reconstitution. The kinetic analysis indicated that the reconstitution reaction proceeds by a sequential mechanism. By a combination of untagged and His-tagged subunits, evidence was obtained for a complex formation between PecE and PecF. Based on the alignments of the lyases and studies of the PecE/F deletion mutants, PecE(1-267) and PecF(57-212) were constructed. Studies showed the activity of PecE(1-267) was not change. The activity of PecF(57-212) was not decreased as the four continuous histidine residues were deleted, which may due to the folding. The four conserved cysteines of PecE(C48Q, C91L, C161L) and PecF(C122L) were constructed. Kinetic studies showed C48 related to the substrate binding, C91 related to the catalysis and the substrate binding; PecF(C122L) nearly lost the isomerization activity, which means C122 located at the active site. PecE and PecF could both bind with PCB, the cysteine residues mutations caused the ability of binding decreased in PecE and an absorption red shift in PecF. Binding PCB could be assayed by Zn2+-induced fluorescence on denaturing gels, illuminated under UV. The absorption changed to a broad range from 550 to 660 nm in acidic urea, and renatured to around 600 nm after dialysis. All deletion mutants constructed could form a complex with its complementary subunits. A single histidine and a tryptophan are required for activity in both PecE and PecF, as judged from diethylpyrocarbonate and N-bromosuccinimide modification and statistical analyses. Inactivation of PecE and PecF is also possible by arginine specific reagents, while modifications of lysine, glutamate and aspartate retained activity. Ten deletion mutants of CpcE/F from the cyanobacterium, Mastigocladus laminosus PCC 7603, were constructed to probe the functional domains. When the N-terminus of CpcE was truncated beyond the R33YYAAWWL motif (CpcE(42-276)) or the C-terminus beyond aa 237, the enzyme lost the ligation activity. The activity decreases sharply to 20% when the C-terminal truncation went beyond L275, which plays a key role for the ligation activity. Enzyme kinetic studies showed that CpcE(L275D) and CpcF(I9K) may acount for the substrate binding. CpcE with PecF could show 6% isomerization activity, which due to the high homology between the E subunits. 20 aa truncation at N-terminus and 39 at C-terminus of CpcE made the lyase lost activity. A 53 aa truncation in CpcF C-terminus caused a loss of the ability to form a 1:1 complex with CpcE, possibly due to misfolding. CpcE and CpcF were subjected to a series of chemical modifications to identify residues essential for catalytic activity. One arginine is essential for CpcE activity, and one tryptophan, and in CpcF one carboxylate. CpcE and CpcF form a 1:1 complex, they also bind PCB tightly, as assayed by Ni2+ chelating affinity chromatography, SDS-PAGE and Zn2+-induced fluorescence. The bound PCB could be transferred to CpcA to yield α-CPC. The PCB transferring capacity could be positively correlated to the activity of the lyase, indicating that PCB bound to CpcE/F can be an intermediate of the enzymatic reaction. A mechanism is proposed for the catalysis, in which CpcE and CpcF interact with each other, bind PCB and adjust via a salt bridge the conformation of PCB, which is then transferred to CpcA.