Nakano Shogo, Kozuka Kohei, Minamino Yuki, Karasuda Hiroka, Hasebe Fumihito, Ito Sohei
Graduate School of Integrated Pharmaceutical and Nutritional Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, 422-8526, Japan.
Commun Chem. 2020 Dec 4;3(1):181. doi: 10.1038/s42004-020-00432-8.
L-amino acid oxidases (LAAOs) can be applied to convert racemic amino acids to D-isomers, which are potential precursors of pharmaceuticals. However, this application is hampered by the lack of available stable and structure-determined LAAOs. In this study, we attempt to address this limitation by utilizing two ancestral LAAOs: AncLAAO-N4 and AncLAAO-N5. AncLAAO-N4 has the highest thermal and temporal stabilities among the designed LAAOs that can be used for deracemization and stereoinversion. AncLAAO-N5 can provide X-ray crystal structures, which are helpful to reveal substrate recognition and reaction mechanisms of LAAOs at the molecular level. Next, we attempted to improve activity of AncLAAO-N4 toward L-Val through a semi-rational protein engineering method. Three variants with enhanced activity toward L-Val were obtained. Taken together, we believe that the activity and substrate selectivity of AncLAAOs give them the potential to be key enzymes in various chemoenzymatic reactions.
L-氨基酸氧化酶（LAAOs）可用于将外消旋氨基酸转化为D-异构体，而D-异构体是药物的潜在前体。然而，由于缺乏可用的稳定且结构明确的LAAOs，这种应用受到了阻碍。在本研究中，我们试图通过利用两种祖先LAAOs：AncLAAO-N4和AncLAAO-N5来解决这一限制。AncLAAO-N4在可用于消旋拆分和立体转化的设计LAAOs中具有最高的热稳定性和时间稳定性。AncLAAO-N5可以提供X射线晶体结构，这有助于在分子水平上揭示LAAOs的底物识别和反应机制。接下来，我们试图通过半理性蛋白质工程方法提高AncLAAO-N4对L-缬氨酸的活性。获得了三种对L-缬氨酸活性增强的变体。综上所述，我们认为AncLAAOs的活性和底物选择性使其有潜力成为各种化学酶促反应中的关键酶。
