Engineering and characterization of hybrid carboxylic acid reductases
Engineering and characterization of hybrid carboxylic acid reductases
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DOI：
10.1016/j.jbiotec.2019.08.008
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发表时间：
2019-10-10
影响因子：
4.1
通讯作者：
Niu, Wei
中科院分区：
文献类型：
--
作者：
Kramer, Levi;Le, Xuan;Niu, Wei
羧酸还原酶（CARs）是有价值的生物催化剂，因为它们能够将广泛的羧酸底物还原成相应的醛产物。car是多结构域酶，具有单独的催化结构域，用于腺苷酸化和随后的底物还原。结构域间动力学对CARs的催化活性至关重要。在这项工作中，基于使用生物信息学和结构分析相结合定义的结构域边界，构建了包含四种细菌CAR和一种真菌CAR结构域的杂交酶。以芳香族和直链（C-3-C-5）羧酸盐为底物，对混合动力汽车进行了稳态和瞬态动力学研究。动力学数据支持域间相互作用在野生型和杂交CAR的功能中发挥重要作用，并进一步导致还原是CAR催化速率决定步骤的假设。我们的研究结果为CAR催化和混合动力CAR工程提供了基本的见解。
Carboxylic acid reductases (CARs) are valuable biocatalysts due to their ability to reduce a broad range of carboxylate substrates into the corresponding aldehyde products. CARs are multi-domain enzymes with separate catalytic domains for the adenylation and the subsequent reduction of substrates. Inter-domain dynamics are crucial for the catalytic activities of CARs. In this work, hybrid enzymes that contain domains from four bacterial CARs and one fungal CAR were constructed based on domain boundaries that were defined using a combination of bioinformatics and structural analysis. Hybrid CARs were characterized in both steady-state and transient kinetics studies using aromatic and straight-chain (C-3-C-5) carboxylate substrates. Kinetic data support that the inter-domain interactions play an important role in the function of both wild-type and hybrid CARs and further lead to the hypothesis that reduction is the rate-determining step in CAR catalysis. Our results provide both fundamental insights into CAR catalysis and rationale for hybrid CAR engineering.