计算设计改造Thermobifida fusca 5-羧基-2-戊烯酰-辅酶A还原酶促进己二酸生产

Computational design of 5-carboxyl-2-pentenoyl-CoA reductase from Thermobifida fusca to enhance adipic acid production

为提高生物合成己二酸的能力,基于计算酶设计对5-羧基-2-戊烯酰-辅酶A还原酶的活性口袋进行改造。基于蛋白质和底物分子结构模型,通过对Ser 88、Leu 89、Ile 90、Pro 91、Ala 92、Val 93、Lys 95、Leu 96、Thr 161、Thr 246、Thr 249、Ile 250、Gln 253和Tyr 367这14个位点进行突变设计,以引入氢键网络来增强突变酶与5-羧基-2-戊烯酰-辅酶A的结合作用,继而提高酶促反应催化效率。在10个设计(Des0~Des9)中,Des0、Des3、Des4和Des9中底物的羧基可与设计的突变Gln253Arg和Ile250Gln形成氢键,且Gln253Arg可与突变Leu89Ser(Des0和Des9)或Leu89Thr(Des3和Des4)以及非设计位点Thr364形成氢键。为检验设计的合理性,该文通过分子动力学模拟分析设计的氢键的稳定性。结果表明,Des0设计中的4个氢键在16 ns分子动力学模拟过程中始终比较稳定,表明Des0可能与5-羧基-2-戊烯酰-辅酶A有较强的结合作用。据此,可推测Des0设计有可能提高催化5-羧基-2-戊烯酰-辅酶A反应的活性,可对其进行后续实验验证。

In order to enhance the biosynthesis of adipic acid,the active pocket of 5-carboxy-2-pentenoyl-CoA reductase was modified using computational enzyme design.Based on the substrate binding model,fourteen residues including Ser 88,Leu 89,Ile 90,Pro 91,Ala 92,Val 93,Lys 95,Leu 96,Thr 161,Thr 246,Thr 249,Ile 250,Gln 253 and Tyr 367 were designed to improve the combination between the enzyme and 5-carboxy-2-pentenoyl-CoA and the catalytic activity of 5-carboxy-2-pentenoyl-CoA reductase by introducing hydrogen bonds network.In the 10 designs(Des0-Des9),the carboxyl of 5-carboxy-2-pentenoyl-CoA could form hydrogen bonds with Gln253Arg and Ile250Gln in Des0,Des3,Des4 and Des9.And Gln253Arg could form hydrogen bonds with Leu89Ser(Des0 and Des9)or Leu89Thr(Des3 and Des4)with Thr364.In order to test these designs,the stability of hydrogen bonds were analyzed by molecular dynamics simulation.The results showed that the four hydrogen bonds designed in Des0 were stable during the process of 16 ns molecular dynamics simulation.It indicated that Des0 may have a strong binding effect with 5-carboxy-2-pentenoyl-CoA.Accordingly,we speculated that Des0 could improve the catalytic activity of 5-carboxy-2-pentenoyl-CoA reductase,which should be verified by subsequent experiments.