Structural and biochemical characterization of fructose‐1,6/sedoheptulose‐1,7–bisphosphatase from the cyanobacterium Synechocystis strain 6803

• Published in: The FEBS journal 2014-02, Vol.281 (3), p.916-926
• Main Authors: Feng, Lingling, Sun, Yao, Deng, Hui, Li, Ding, Wan, Jian, Wang, Xiaofeng, Wang, Weiwei, Liao, Xun, Ren, Yanliang, Hu, Xiaopeng
• Format: Article
• Language: English
• Subjects: Adenosine Monophosphate - antagonists & inhibitors; Adenosine Monophosphate - chemistry; Adenosine Monophosphate - metabolism; Algae; Allosteric Regulation; Bacterial Proteins - antagonists & inhibitors; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Binding Sites; Biocatalysis; biochemical characterization; Biochemistry; Catalysis; Circular Dichroism; Cyanobacteria; Enzymes; Fructose-Bisphosphatase - antagonists & inhibitors; Fructose-Bisphosphatase - chemistry; Fructose-Bisphosphatase - genetics; Fructose-Bisphosphatase - metabolism; Fructosediphosphates - chemistry; Fructosediphosphates - metabolism; fructose‐1,6/sedoheptulose‐1,7‐bisphosphatase, site‐directed mutagenesis; Kinetics; Ligands; Models, Molecular; Molecular Conformation; Molecular Dynamics Simulation; Mutagenesis, Site-Directed; Mutant Proteins - antagonists & inhibitors; Mutant Proteins - chemistry; Mutant Proteins - metabolism; Phosphoric Monoester Hydrolases - antagonists & inhibitors; Phosphoric Monoester Hydrolases - chemistry; Phosphoric Monoester Hydrolases - genetics; Phosphoric Monoester Hydrolases - metabolism; Protein Subunits - antagonists & inhibitors; Protein Subunits - chemistry; Protein Subunits - genetics; Protein Subunits - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; structure; Synechocystis; Synechocystis - enzymology; X-Ray Diffraction
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/febs.12657

Cyanobacterial fructose–1,6/sedoheptulose‐1,7–bisphosphatase (cy–FBP/SBPase) plays a vital role in gluconeogenesis and in the photosynthetic carbon reduction pathway, and is thus a potential enzymatic target for inhibition of harmful cyanobacterial blooms. Here, we describe the crystal structure of cy–FBP/SBPase in complex with AMP and fructose‐1,6–bisphosphate (FBP). The allosteric inhibitor AMP and the substrate FBP exhibit an unusual binding mode when in complex with cy–FBP/SBPase. Binding mode analysis suggested that AMP bound to the allosteric sites near the interface across the up/down subunit pairs C1C4 and C2C3 in the center of the tetramer, while FBP binds opposite to the interface between the horizontal subunit pairs C1C2 or C3C4. We identified a series of residues important for FBP and AMP binding, and suggest formation of a disulfide linkage between Cys75 and Cys99. Further analysis indicates that cy–FBP/SBPase may be regulated through ligand binding and alteration of the structure of the enzyme complex. The interactions between ligands and cy–FBP/SBPase are different from those of ligand‐bound structures of other FBPase family members, and thus provide new insight into the molecular mechanisms of structure and catalysis of cy–FBP/SBPase. Our studies provide insight into the evolution of this enzyme family, and may help in the design of inhibitors aimed at preventing toxic cyanobacterial blooms. Database Structural data have ben submitted to the Protein Data Bank under accession numbers 3ROJ and 3RPL. Structured digital cy-FBP/SBPase and cy-FBP/SBPase bind by x-ray crystallography (View interaction). We describe the crystal structure of cy‐FBP/SBPase +AMP+FBP, which is dynamic and harbors at least three regulatory domains. The state of cy‐FBP/SBPase is changed among three potential states in the presence of different regulators: initial non‐active dimer state, non‐active tetramer state and active tetramer state. In the non‐active dimer state, a disulfide bridge can be formed between Cys75 and Cys99.