Masahide Kawamoto

Inorganic pyrophosphatases are essential metalloenzymes for phosphate metabolism. Bacterial Family II Inorganic pyrophosphatases utilize a trinuclear metal center and exhibit higher catalytic activity than binuclear counterparts. Here we show the mechanism underlying this enhanced hydrolytic efficiency in the enzyme from Shewanella sp. AS-11 using X-ray absorption spectroscopy, site-directed mutagenesis, and density functional theory calculations. We identify a catalytic μ ₃ -oxo nucleophile—generated by proton transfer from a bridging μ ₃ -hydroxide to Asp14—as the key reactive species for hydrolysis. Rotation of Asp14 drives this conversion and constitutes the rate-limiting step, with an activation barrier of 15.5 kcal mol ^-1 . The trinuclear metal center promotes hydrolysis by lowering the p K _a of the hydroxide to facilitate μ ₃ -oxo formation, stabilizing this intermediate, positioning the nucleophile for optimal in-line attack, and enhancing phosphorus electrophilicity. These findings highlight the importance of reactive species generation and illustrate how metalloenzymes exploit geometric and electronic tuning to achieve high catalytic reactivity.

A lens-coupled high-speed X-ray camera, “Hayaka”, was developed for quick imaging of X-ray absorption fine structure (XAFS) and time-resolved high-speed computed tomography (CT) using synchrotron radiation (SR). This camera is a lens-coupled type, composed of a scintillator, an imaging lens system, and a high-speed visible light sCMOS, capable of imaging with a minimum exposure time of 1 μs and a maximum frame rate of 5000 frames/s (fps). A feasibility study using white and monochromatic SR at the beamline BL07 of the SAGA Light Source showed that fine X-ray images with a spatial resolution of 77 μm can be captured with an exposure time of 10 μs. Furthermore, quick imaging XAFS, combined with high-speed energy scanning of a small Ge double crystal monochromator of the same beamline, enabled spectral image data to be acquired near the Cu K-edge in a minimum of 0.5 s. Additionally, an in coquendo 4DCT (time-resolved 3D observation of cooking processes) observation combined with a high-speed rotation table revealed the boiling process of Japanese somen noodles over 150 s with a time resolution of 0.5 s.

Sulfur K-edge XAFS measurements were performed to evaluate the molecular orientation of DNTT thin films and the local structure of DBTTT films. A new tender XAFS measurement system was developed and installed at beamline BL11 of the SAGA Light Source to enable reliable and high-throughput structural analysis of sulfur-containing organic semiconductor thin films and related materials. The system supports rapid, automated angle-dependent measurements using a motorized sample manipulator under a helium atmosphere (1 atm), without requiring vacuum. It is equipped with a conversion electron yield (CEY) electrode and a silicon drift detector (SDD) for partial fluorescence yield (PFY), enabling simultaneous detection with different surface sensitivities. Samples can be introduced without air exposure via a transfer vessel and load-lock chamber, allowing exchange in less than 5 minutes and acquisition of high-quality spectra in about 6 minutes. The new system contributes to improving the analytical capabilities for such materials.

Efficient catalysis by metalloproteins relies on precise spatial arrangement of metal ions and active-site residues. Family II inorganic pyrophosphatase (PPase) from Shewanella species features a trinuclear metal center and displays higher catalytic activity than binuclear counterparts. Here we elucidate its hydrolytic mechanism using X-ray crystal structure-based extended X-ray absorption fine structure (XCS-EXAFS), site-directed mutagenesis, and density functional theory (DFT) calculations. We identify a catalytic μ₃-oxo nucleophile, formed via proton transfer from a bridging μ₃-hydroxide to Asp14 and subsequent hydrogen-bond rearrangement to Asp72, as the key species in S _N 2-type hydrolysis. This conversion defines the rate-limiting step with an activation barrier of 15.5 kcal/mol. Molecular orbital analysis reveals that the trinuclear cluster promotes μ₃-oxo formation, aligns the nucleophile for attack, and stabilizes the transition state. The side-chain rotation of the conserved Asp14 is crucial for catalysis. Our results highlight how metalloenzymes exploit geometric and electronic tuning to achieve high reactivity through evolutionarily optimized architectures.

Developing highly efficient methane (CH4) hydrate storage methods and understanding the hydrate dissociation kinetics can contribute to advancing CH4 gas storage and transport. The effects of tetrabutylammonium bromide (TBAB) (a thermodynamic promoter) addition on the kinetics of CH4 hydrate were evaluated on the microscopic scale using synchrotron x-ray computed tomography (CT) and powder x-ray diffraction. Microscopic observations showed that a 5 wt. % TBAB solution facilitated the nucleation of CH4 hydrate owing to the initial growth of TBAB semi-clathrate hydrate particles. The CH4 hydrate crystals in the CH4 + TBAB hydrate sample were sponge-like with many internal pores and exhibited slightly enhanced self-preservation compared to the pure CH4 hydrate, both in the bulk and after pulverization to a fine powder. This study demonstrates the feasibility of controlling the rate of CH4 hydrate formation and preservation by using aqueous TBAB solutions in CH4 hydrate formation.

The SAGA Light Source provides X-ray imaging resources based on high-intensity synchrotron radiation (SR) emitted from the superconducting wiggler at beamline 07 (BL07). By combining quasi-monochromatic SR obtained by the newly installed water-cooled metal filter and monochromatic SR selected by a Ge double-crystal monochromator (DCM) with high-resolution lens-coupled X-ray imagers, fast and low-dose micro-computed tomography (CT), fast phase-contrast CT using grating-based X-ray interferometry, and 2D micro-X-ray absorption fine structure analysis can be performed. In addition, by combining monochromatic SR obtained by a Si DCM with large-area fiber-coupled X-ray imagers, high-sensitivity phase-contrast CT using crystal-based X-ray interferometry can be performed. Low-temperature CT can be performed using the newly installed cryogenic system, and time-resolved analysis of the crystallinity of semiconductor devices in operation can be performed using a time-resolved topography system. The details of each instrument and imaging method, together with exemplary measurements, are presented.

Diffraction-enhanced imaging (DEI) has high sensitivity and a wide dynamic range of density and thus can be used for fine imaging of biological and organic samples that include large differences in density. A fast DEI method composed of continuous fast sample rotations and slow analyzer crystal scanning was developed to shorten the measurement period. Fine sectional images of a biological sample were successfully obtained within a half measurement period of the conventional step-scanning method while keeping the same exposure time. In addition, a fine three-dimensional image of a rat tail was obtained with a 375 s measurement period.