Structures And Enzymatic Mechanisms Of Phycobiliprotein Lyases CpcE/F And PecE/F

The light-harvesting phycobilisome in cyanobacteria and red algae requires the lyase-catalyzed chromophorylation of phycobiliproteins.There are three functionally distinct lyase families known.S/U-type lyases ligate phycocyanobilin to the Cys81 site of allophycocyanins and the Cys84 site of ?-subunits in phycocyanins,phycoerythrins and phycoerythrocyanins.T-type lyases attach phycocyanobilin to the Cys153 site of ?-subunits in phycocyanins and phycoerythrocyanins.The heterodimeric E/F-type lyases are specific for attaching phycocyanobilin to the Cys84 site of ?-subunits in phycocyanins and phycoerythrocyanins.Unlike other lyases,the E/F-type lyases also have chromophore-detaching activity.A subclass of the E/F-type lyases is,furthermore,capable of chemically modifying the chromophore.The crystal structures of an S-type lyase,CpcS,and a T-type lyase,CpcT,have been determined with or without the chromophore.They both adopt a ?-barrel structure similar to those of fatty acid binding proteins.Although E/F-type lyases were characterized >25 y ago,their structures remained unknown.The work determined the crystal structure of the heterodimer of CpcE/F from Nostoc sp.PCC7120 at 1.89 ? resolution.Both subunits are twisted,crescent-shaped ?-solenoid structures.CpcE has 15 and CpcF 10 helices.The inner(concave)layer of CpcE and the outer(convex)layer of CpcF form a cavity into which the phycocyanobilin chromophore can be modeled.This location of the chromophore is supported by mutations at the interface between the subunits and within the cavity.The structure of a structurally related,isomerizing lyase,PecE/F,that converts phycocyanobilin into phycoviolobilin,was modeled using the CpcE/F structure as template.A H87C88 motif critical for the isomerase activity of PecE/F is located at the loop between h20 and h21,supporting the proposal that the nucleophilic addition of Cys88 to C10 of phycocyanobilin induces the isomerization of phycocyanobilin into phycoviolobilin.Also,the structure of NblB,involved in phycobilisome degradation could be modeled using CpcE as template.To obtain more information on mechanism,docking complexs are modeled between the two lyases,CpcE/F and PecE/F,and their substrates.According these docking models,the binding of Cys84 and chromophore would require transient loosening of the helix-helix interaction of phycobiliprotein to allow transfer of the chromophore to the binding site.CpcE/F cannot only ligate phycocyanobilin to apo-CpcA,but also detach it from the holoprotein,PCB-CpcA.Considering the reversibility of the reaction,a hypothesis has been proposed that the chromophore moves in different direction during chromophorylation and chromophore-detaching.The PCB chromophore moves during chromophorylation more in the direction of CpcE,as CpcA contacts mainly with CpcE,and thus this movement will facilitate PCB attachment to CpcA.Conversely,during the detachment of PCB from PCB-CpcA,the bound PCB chromophore might be attracted to CpcF and finally complexed with CpcF,thus facilitating PCB detachment.Hindrance of this movement by the unstructured N-terminal arm of CpcE located in front of CpcF would explain the enhanced detachment by truncation of this arm.The first crystal structure of E/F-type lyases was determined.The work fills in a missing structural and mechanistic link for our understanding of the bilin lyase enzyme families and how these important enzymes function to modify the entire light harvesting apparatus present in this critical group of oxygenic phototrophic bacteria.In addition,this enzyme is the only one that has bilin removal activity,presumably as a way to begin the degradation process of these proteins during nutrient limitation or as a first step in remodeling of the phycobilisome that occurs during chromatic acclimation.