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. DatabaseStructural data have ben submitted to the Protein Data Bank under accession numbers 3ROJ and 3RPL.Structured digital abstract cy-FBP/SBPase and cy-FBP/SBPase bind by x-ray crystallography (View interaction).

The optical characteristics of absorption and circular dichroism (CD) spectroscopy of an alpha-subunit of C-phycocyanin (C-PC) were investigated by using time dependent density functional theory (TDDFT) combined with the polarizable continuum model (PCM). When the protonation of alpha-84 phycocyanobilin (PCB) and its interaction with the protein moiety in C-PC have been taken into account, satisfactory assignment of the absorption and CD spectra of alpha-84 PCB can be achieved. The TDDFT-PCM calculations conclude that in the visible absorption region the main peak arises from the pi electron excitation of the pyrrole rings and the shoulder peak comes from the charge transfer from Asp87 (a nearby amino acid residue) to PCBH(+).

By using time-dependent density functional theory combined with the polarizable continuum model, a satisfactory assignment of the absorption and circular dichroism spectra and energy transfer flow of the beta-subunit in C-phycocyanin (C-PC) was achieved when the protonation of beta-84 and beta-155 phycocyanobilin (PCB) and their interaction with the protein moiety in C-PC have been taken into account. We attribute the main peak for both beta-84 and beta-155 as arising from the pi electron excitation of the pyrrole rings and the shoulder peak as arising from the charge transfer from the asparate residue to PCBH(+). The satisfactory agreement between theory and experiment suggests that Förster resonance theory prevails such that energy transfer occurs from beta(s) (beta-155) to beta(f) (beta-84).

A handful of molecular docking tools have been extended to enable a covalent docking. However, all of them face the challenge brought by the covalent bond between proteins and ligands. Many covalent drug design scenarios still heavily rely on demanding crystallographic experiments for accurate binding structures. Aiming at filling the gap between covalent dockings and crystallographic experiments, we develop and validate a hybrid method, dubbed as Cov_DOX, in this work. Cov_DOX achieves an overall success rate of 81% with RMSD < 2 Å for the Top 1 pose prediction in the validation against a test set including 405 crystal structures for covalent protein–ligand complexes, covering various types of the warhead chemistry and receptors. Such accuracy is not far from the much more demanding crystallographic experiments, in sharp contrast to the performance of the covalent docking front runners (success rate: 40–60%).

Class II fructose-1,6-bisphosphate aldolases (FBA-II) are attractive new targets for the discovery of drugs to combat invasive fungal infection, because they are absent in animals and higher plants. Although several FBA-II inhibitors have been reported, none of these inhibitors exhibit antifungal effect so far. In this study, several novel inhibitors of FBA-II from C. albicans (Ca-FBA-II) with potent antifungal effects were rationally designed by jointly using a specific protocols of molecular docking-based virtual screening, accurate binding-conformation evaluation strategy, synthesis and enzymatic assays. The enzymatic assays reveal that the compounds 3c, 3e-g, 3j and 3k exhibit high inhibitory activity against Ca-FBA-II (IC < 10 μM), and the most potential inhibitor is 3g, with IC value of 2.7 μM. Importantly, the compounds 3f, 3g, and 3l possess not only high inhibitions against Ca-FBA-II, but also moderate antifungal activities against C. glabrata (MIC = 4-64 μg/mL). The compounds 3g, 3l, and 3k in combination with fluconazole (8 μg/mL) displayed significantly synergistic antifungal activities (MIC < 0.0625 μg/mL) against resistant Candida strains, which are resistant to azoles drugs. The probable binding modes between 3g and the active site of Ca-FBA-II have been proposed by using the DOX (docking, ONIOM, and XO) strategy. To our knowledge, no FBA-II inhibitors with antifungal activities against wild type and resistant strains from Candida were reported previously. The positive results suggest that the strategy adopted in this study are a promising method for the discovery of novel drugs against azole-resistant fungal pathogens in the future.