Biology subjects

Osawa, Y.

Publications and source records attributed to Osawa, Y..

4 recordsLinked to original sources

bioRxiv2026

Structural Basis of Electron Transfer by the Human Nitric Oxide Synthase Holoenzyme Complex

Mammalian nitric oxide synthase (NOS) generates nitric oxide (NO), an essential signaling molecule in neurotransmission, inflammation, and cardiovascular regulation. NOS dysregulation contributes to neurodegeneration, septic shock, and ischemia. The structural basis of NOS function remains unclear, likely due to its dynamic, multi-component architecture. Catalysis requires electron transfer across five cofactors, with the oxygenase (Oxy) domain converting L-arginine to NO after receiving electrons from the reductase (Red) domain via calmodulin (CaM) activation. Here, we report cryo-electron microscopy structures of the human inducible NOS:CaM holoenzyme, revealing a previously unresolved architecture in which the Red:CaM arm spans the Oxy dimer in an apparent active state for cross-monomer electron transfer. The FMN subdomain is rotated [~]90{degrees} relative to the Red core, positioning its cofactor [~]9 [A] from the heme, adjacent to a conserved aromatic residue within a narrow Oxy domain tunnel. Red-Oxy flexibility in the active state may facilitate electron shuttling among cofactors, defining how NOS cofactors are transiently aligned to enable catalysis.

biochemistry↗

bioRxiv2025

Computational design of CYP102A1 variants for the biosynthesis of a next-generation antiplatelet drug DT-678

Clopidogrel is a widely used antiplatelet prodrug to treat acute coronary syndromes. However, its clinical efficacy is hampered by ineffective bioactivation to produce the pharmacologically active metabolite (AM), leading to variability in antiplatelet response among different ethnic groups. To overcome the shortcomings of clopidogrel, DT-678 was developed by conjugating the AM to 3-nitropyridine-2-thiol via a mixed disulfide bond. It has been challenging to produce the conjugate in high yield by chemical synthesis. Here, we report the first de novo biosynthesis of DT-678 using engineered CYP102A1 variants. We applied structure-based computational design using UniDesign to generate three variants (UD4, UD5, and UD6) that enhanced the catalytic activity and selectivity toward DT-678 synthesis. Among them, UD6 demonstrated the highest total turnover number and DT-678-specific productivity under optimized conditions. Mechanistic analysis revealed that rapid enzyme inactivation, driven by reactive oxygen species (ROS) such as superoxide and hydrogen peroxide, limited overall yield. Remarkably, we found that ascorbic acid significantly protected CYP102A1 variants from inactivation and hence increased production yield. This work establishes a scalable enzymatic strategy for DT-678 biosynthesis and highlights the importance of combining protein engineering with redox control to overcome limitations in CYP-catalyzed reactions.

biochemistry↗

bioRxiv2021

Production of offspring from azoospermic mice with meiotic failure: Precise biparental meiosis within halved oocytes

While the large volume of mammalian oocytes is necessary for embryo development, it can lead to error-prone chromosomal segregation during meiosis. Conversely, we hypothesized that smaller oocytes would have a great unidentified potential to stabilize unstable meiosis and improve the development of the resultant embryos. Here, we show that reducing ooplasmic volume can rescue highly error-prone fertilization using primary spermatocytes by preventing segregation errors of chromosomes during biparental meiosis. High-resolution live-imaging analysis revealed that erroneous chromosome segregation occurred in most (90%) spermatocyte-injected oocytes of normal size, but could be ameliorated to 40% in halved oocytes. The birth rate improved remarkably from 1% to 19% (P < 0.0001). Importantly, this technique enabled the production of offspring from azoospermic mice with spermatocyte arrest caused by STX2 deficiency, an azoospermia factor also found in humans. Thus, contrary to popular opinion, oocytes inherently have a strong potential for precise meiotic divisions, which can be evoked by reduction of the ooplasmic volume. Their potential might help rescue cases of untreatable human azoospermia with spermatocyte arrest.

developmental biology↗

bioRxiv2020

EXOC1 regulates cell morphology of spermatogonia and spermatocytes in mice

Spermatogenesis requires high regulation of germ cell morphology. The spermatogonia regulates its differentiation state by its own migration. The male germ cells differentiate and mature with the formation of syncytia, failure of forming the appropriate syncytia results in the arrest of spermatogenesis at the spermatocyte stage. However, the detailed molecular mechanisms of male germ cell morphological regulation are unknown. Here, we found that EXOC1 is important for the pseudopod formation of spermatogonia and spermatocyte syncytia in mice. We found that while EXOC1 contributes to the inactivation of Rac1 in the pseudopod formation of spermatogonia, in spermatocyte syncytium formation, EXOC1 and SNAP23 cooperate with STX2. Our results showed that EXOC1 functions in concert with various cell morphology regulators in spermatogenesis. Since EXOC1 is known to bind to several cell morphogenesis factors, this study is expected to be the starting point for the discovery of many morphological regulators of male germ cells.

developmental biology↗