Liang Chaoqun, Duan Xiuyuan, Shahab Muhammad, Zheng Guojun
State Key Laboratory of Chemical Resources Engineering, Beijing University of Chemical Technology, Beijing 100029, China.
State Key Laboratory of Chemical Resources Engineering, Beijing University of Chemical Technology, Beijing 100029, China.
J Biosci Bioeng. 2023 May;135(5):369-374. doi: 10.1016/j.jbiosc.2023.02.009. Epub 2023 Mar 17.
(S)-4-(Hydroxymethyl)cyclopent-2-enone is a key intermediate in the synthesis of chiral five-membered carbasugars, which can be used to synthesize a large number of pharmacologically relevant carbocyclic nucleosides. Herein, CV2025 ω-transaminase from Chromobacterium violaceum was selected based on substrate similarity to convert ((1S,4R)-4-aminocyclopent-2-enyl)methanol to (S)-4-(hydroxymethyl)cyclopent-2-enone. The enzyme was successfully cloned, expressed in Escherichia coli, purified and characterized. We show that it has R configuration preference in contrast with the conventional S preference. The highest activity was obtained below 60 °C and at pH 7.5. Cations Ca and K enhanced activity by 21% and 13%, respectively. The conversion rate reached 72.4% within 60 min at 50 °C, pH 7.5, using 0.5 mM pyridoxal-5'-phosphate, 0.6 μM CV2025, and 10 mM substrate. The present study provides a promising strategy for preparing five-membered carbasugars economically and efficiently.
（S）-4-（羟甲基）环戊-2-烯酮是合成手性五元碳环糖的关键中间体，可用于合成大量具有药理活性的碳环核苷。在此，基于底物相似性选择了来自紫色杆菌的CV2025 ω-转氨酶，将（（1S，4R）-4-氨基环戊-2-烯基）甲醇转化为（S）-4-（羟甲基）环戊-2-烯酮。该酶成功克隆，在大肠杆菌中表达、纯化并进行了表征。我们发现它与传统的S偏好相反，具有R构型偏好。在60°C以下和pH 7.5时活性最高。阳离子Ca和K分别使活性提高了21%和13%。在50°C、pH 7.5条件下，使用0.5 mM磷酸吡哆醛、0.6 μM CV2025和10 mM底物，60分钟内转化率达到72.4%。本研究为经济高效地制备五元碳环糖提供了一种有前景的策略。
