Open Gate of Corynebacterium glutamicum Threonine Deaminase for Efficient Synthesis of Bulky α-Keto Acids
Open Gate of Corynebacterium glutamicum Threonine Deaminase for Efficient Synthesis of Bulky α-Keto Acids
复制标题
打开谷氨酸棒杆菌苏氨酸脱氨酶之门，有效合成大体积α-酮酸
DOI：
10.1021/acscatal.0c01672
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发表时间：
2020-09-04
期刊：
影响因子：
12.9
通讯作者：
Liu, Liming
中科院分区：
文献类型：
--
作者：
Song, Wei;Xu, Xin;Liu, Liming
Threonine deaminase (TD, EC 4.3.1.19) mediates alpha,beta-elimination (deamination), which has untapped potential in the synthesis of unnatural alpha-keto acids. However, the narrow substrate scope of wild-type TD limits its application. This issue could be overcome by engineering the substrate tunnel of the enzyme. Here, Corynebacterium glutamicum TD (CgTD) was used as a model, and its substrate tunnel was identified as a gating element. On the basis of the gating characteristics and constitution of the substrate access tunnel of CgTD, an "open-gate" strategy was developed to improve its substrate scope toward bulky substrates. The best variant obtained following this approach, CgTD(Mu7), exhibited 90.6-fold greater catalytic efficiency (k(cat)/K-M) toward a bulky substrate such as phenylserine. The improvement was due to more efficient substrate coupling, deprotonation, and imine hydrolysis. CgTD(Mu7) was used for efficient production of both natural and unnatural a-keto acids, including 2-oxobutyric acid (83.4 g L-1, 99% conversion), phenylpyruvic acid (72.5 g L-1, 95% conversion), 2-oxovaleric acid (21.1 g L-1, 91% conversion), and 2-oxo-4-phenylbutyric acid (29.9 g L-1, 84% conversion). The proposed strategy provides a theoretical basis for the synthesis of a-keto acids and is also effective for engineering other similar enzymes.
