Fang Yu, Zhang Shuyan, Wang Jianli, Yin Lianghong, Zhang Hailing, Wang Zhen, Song Jie, Hu Xiaoqing, Wang Xiaoyuan
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China.
Zhejiang Provincial Key Laboratory of Resources Protection and Innovation of Traditional Chinese Medicine, Zhejiang A&F University, Hangzhou 311300, China.
Metabolites. 2021 Jan 4;11(1):30. doi: 10.3390/metabo11010030.
2-Oxobutyrate (2-OBA), as a toxic metabolic intermediate, generally arrests the cell growth of most microorganisms and blocks the biosynthesis of target metabolites. In this study, we demonstrated that using the acetate bypass to replace the pyruvate dehydrogenase complex (PDHc) in could recharge the intracellular acetyl-CoA pool to alleviate the metabolic toxicity of 2-OBA. Furthermore, based on the crystal structure of pyruvate oxidase (PoxB), two candidate residues in the substrate-binding pocket of PoxB were predicted by computational simulation. Site-directed saturation mutagenesis was performed to attenuate 2-OBA-binding affinity, and one of the variants, PoxB, exhibited a 20-fold activity ratio of pyruvate/2-OBA in substrate selectivity. PoxB was employed to remodel the acetate bypass in , resulting in l-threonine (a precursor of 2-OBA) biosynthesis with minimal inhibition from 2-OBA. After metabolic detoxification of 2-OBA, the supplies of intracellular acetyl-CoA and NADPH (nicotinamide adenine dinucleotide phosphate) used for l-threonine biosynthesis were restored. Therefore, 2-OBA is the substitute for pyruvate to engage in enzymatic reactions and disturbs pyruvate metabolism. Our study makes a straightforward explanation of the 2-OBA toxicity mechanism and gives an effective approach for its metabolic detoxification.
2-氧代丁酸（2-OBA）作为一种有毒的代谢中间体，通常会抑制大多数微生物的细胞生长，并阻断目标代谢物的生物合成。在本研究中，我们证明，在[具体内容缺失]中利用乙酸旁路替代丙酮酸脱氢酶复合物（PDHc）可以补充细胞内乙酰辅酶A库，以减轻2-OBA的代谢毒性。此外，基于丙酮酸氧化酶（PoxB）的晶体结构，通过计算模拟预测了PoxB底物结合口袋中的两个候选残基。进行定点饱和诱变以减弱2-OBA结合亲和力，其中一个变体PoxB在底物选择性方面表现出丙酮酸/2-OBA 20倍的活性比。利用PoxB对[具体内容缺失]中的乙酸旁路进行重塑，从而在2-OBA抑制作用最小的情况下合成L-苏氨酸（2-OBA的前体）。在对2-OBA进行代谢解毒后，用于L-苏氨酸生物合成的细胞内乙酰辅酶A和烟酰胺腺嘌呤二核苷酸磷酸（NADPH）供应得以恢复。因此，2-OBA替代丙酮酸参与酶促反应并扰乱丙酮酸代谢。我们的研究对2-OBA毒性机制做出了直接解释，并为其代谢解毒提供了有效方法。
