Carbonic anhydrase (CA) catalyzes the reversible hydration of carbon dioxide (CO2) to bicarbonate (HCO3 -) and plays an essential role in carbon fixation in marine diatoms. Here we report the structural and functional characterization of a novel CA, θ-CA3, from the diatom Phaeodactylum tricornutum, elucidating its physiological role and catalytic mechanism. AlphaFold prediction, sequence alignment, and metal analysis showed that θ-CA3 is a dimeric enzyme, with each monomer composed of two zinc-binding catalytic domains. High-resolution X-ray crystallographic structures of domain 2 of θ-CA3 in the CO2-bound form revealed the detailed substrate binding pattern in the active site. Site-directed mutagenesis showed that Asp49 and Arg117 in the active site are essential for catalysis. Notably, introducing a negative charge near the active-site entrance resulted in a mutant enzyme with markedly increased activity under acidic pH, suggesting that electrostatic modulation of the active-site environment regulates proton transfer and catalysis. Furthermore, we identified an HCO3 - ion at the dimer interface that contributes to enzyme activation. Collectively, our findings provide fundamental structural insight into how the active-site electrostatic charges and metal environment govern the catalytic efficiency of θ-CA3, offering a new perspective on the molecular basis of carbon fixation in diatoms.
Hiroto Negoro, Atsuki Ohsawa, Ginga Shimakawa et al.· The FEBS Journal· 0 citations
Three-dimensional covalent organic frameworks (3D COFs) are promising crystalline porous materials, but the elucidation of their structure remains challenging, particularly for those featuring spiroborate linkages. Herein, we report the synthesis of a 3D crystalline COF with nbo topology, constructed from a rigid square-planar monomer, tetracyclopentatetraphenylene (TCTP), and spiroborate linkages. Theoretical calculations revealed that the TCTP core has higher rigidity than phthalocyanine, effectively suppressing structural fluctuations during framework formation. The structure of the resulting TCTP-COF was successfully determined using microcrystal electron diffraction (MicroED), revealing a noninterpenetrated cubic framework. TCTP-COF exhibits high crystallinity, thermal stability up to 320°C, and permanent porosity with a Brunauer-Emmett-Teller surface area of 1360 square meters per gram. This work represents the structural determination of a spiroborate-linked 3D crystalline COF using MicroED methods, providing a design strategy for expanding the chemical space of highly ordered 3D COF architectures.
Protein Data Bank Japan (https://pdbj.org/) is the Asian hub of three‐dimensional (3D) macromolecular structure data and a founding member of the global Protein Data Bank (PDB) network. Over two decades, we have curated and distributed experimentally determined structures, complementing international collaborations with Research Collaboratory for Structural Bioinformatics (RCSB) PDB, Biological Magnetic Resonance Data Bank, Protein Data Bank in Europe (PDBe), and Electron Microscopy Data Bank. In response to user demand for integrated structural and chemical data, we developed a new PubChem Portal that enables interactive exploration of compound‐protein interactions. Users can view ligand binding poses in 3D via our Web Graphics Library (WebGL)‐based Molmil viewer, with key interactions highlighted and key residues displayed in semi‐transparent stick models, enhanced through integration with secondary databases (e.g., Dynamics DB, eF‐site) for advanced insights into molecular dynamics and electrostatics. The system supports filtering by UniProt ID, Enzyme Commission (EC) number, Pfam ID, or PROSITE ID to identify structurally related compounds and visualizes protein–ligand interactions. A dynamic two‐dimensional (2D) Japan Agency for Medical Research and Development representation enables real‐time atom‐level navigation, with clickable atoms linking to 3D structures. This tool allows users to explore compound‐protein interaction landscapes, identify potential binding modes, and guide experimental design, such as mutagenesis or crystallization. The portal offers a comprehensive, user‐centered ecosystem that bridges chemical and structural data, enhancing access to biological insights through integrated visualization and analysis.
G. Bekker, Chioko Nagao, Satomi Niwa et al.· Protein Science· 1 citation