化学
突变体
饱和突变
立体化学
氟化物
酶
选择性
活动站点
生物化学
催化作用
无机化学
基因
作者
Yixun Jiang,Minyu Yao,Hongxing Niu,Wenrui Wang,Jie He,Bin Qiao,Bingzhi Li,Min Dong,Wenhai Xiao,Ying‐Jin Yuan
标识
DOI:10.1021/acs.jafc.3c08185
摘要
Organofluorine compounds have attracted substantial attention owing to their wide application in agrochemistry. Fluorinase (FlA) is a unique enzyme in nature that can incorporate fluorine into an organic molecule. Chlorinase (SalL) has a similar mechanism as fluorinase and can use chloride but not fluoride as a substrate to generate 5′-chloro-deoxyadenosine (5′-ClDA) from S-adenosyl-l-methionine (SAM). Therefore, identifying the features that lead to this selectivity for halide ions is highly important. Here, we engineered SalL to gain the function of FlA. We found that residue Tyr70 plays a key role in this conversion through alanine scanning. Site-saturation mutagenesis experiments demonstrated that Y70A/C/S/T/G all exhibited obvious fluorinase activity. The G131S mutant of SalL, in which the previously thought crucial residue Ser158 for fluoride binding in FlA was introduced, did not exhibit fluorination activity. Compared with the Y70T single mutant, the double mutant Y70T/W129F increased 5′-fluoro-5-deoxyadenosine (5′-FDA) production by 76%. The quantum mechanics (QM)/molecular mechanics (MM) calculations suggested that the lower energy barriers and shorter nucleophilic distance from F– to SAM in the mutants than in the SalL wild-type may contribute to the activity. Therefore, our study not only renders SalL the activity of FlA but also sheds light on the enzyme selectivity between fluoride versus chloride.
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