5-氨基乙酰丙酸合成酶(5-aminolevulinic acid synthase,ALAS)是α-氧代胺合酶家族中的一种磷酸吡哆醛依赖性脱羧酶,能够催化甘氨酸和琥珀酰-辅酶A生成5-氨基乙酰丙酸(5-aminolevulinic acid,5-ALA)。然而,大多数ALAS存在活性较低和热稳定性差的问题,限制了其在工业化生产中的应用。该研究对沼泽红假单胞菌(Rhodopseudomonas palustris)来源的RpALAS进行了分子改造,旨在提高其催化活性和热稳定性。通过利用Fireprot和HotSpot Wizard在线工具设计突变体,成功获得了一株热稳定性显著提升的突变体M4(T73L/L252F/T396M)。该突变体不仅酶活力提高了5%,最适反应温度提升了8 ℃,最适pH值也向中性偏移了0.5个单位。突变体M4在45 ℃下的半衰期(t1/2)达到214.43 min,是野生型的8.9倍。分子动力学模拟分析表明,蛋白质疏水作用的增强和空间位阻的变化是导致酶热稳定性提升的关键因素。该研究成功获得了具有优良热稳定性的ALAS突变体,不仅丰富了ALAS突变体酶库,而且为深入理解ALAS结构与功能的关系提供了重要的实验依据和理论参考。
5-Aminolevulinic acid synthase (5-ALAS), a pyridoxal phosphate-dependent decarboxylase within the α-oxoamine synthase family, catalyzes the condensation of glycine and succinyl-CoA to produce 5-aminolevulinic acid (5-ALA).However, the low activity and poor thermal stability of most ALAS enzymes hinder their application in industrial production.In this study, molecular engineering was conducted on RpALAS derived from Rhodopseudomonas palustris to enhance its catalytic activity and thermal stability.Through rational design using the online tools Fireprot and HotSpot Wizard, this study successfully developed a mutant enzyme, M4 (T73L/L252F/T396M), which demonstrated remarkable improvements in thermal stability.This mutant not only exhibited a 5% increase in enzyme activity but also had an optimal reaction temperature that was 8 ℃ higher and an optimal pH that shifted 0.5 units towards neutrality compared to the wild type.The half-life (t1/2) of mutant M4 at 45 ℃ reached 214.43 minutes, which was 8.9 times that of the wild type.Molecular dynamics simulation analysis revealed that the enhanced hydrophobic interactions and changes in steric hindrance within the protein were key factors contributing to the improved thermal stability of the enzyme.This study successfully obtained an ALAS mutant with excellent thermal stability, which not only enriched the library of ALAS mutants but also provided important experimental evidence and theoretical references for a deeper understanding of the relationship between ALAS structure and function.
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