L-天冬氨酸-α-脱羧酶(PanD)是生物体中催化L-天冬氨酸脱羧成β-丙氨酸的必需酶。为了执行催化功能,PanD前蛋白需要自我切割以形成两个亚基:活性α亚基和β亚基。然而,由于未知的原因,在不同的生物体中,自我分裂的过程已经不同。为了揭示可能的分歧机制,分子进化,本研究探讨了panD基因家族的选择压力和定点诱变。进化分析表明,细菌中的panD基因分为三个分支:I类,二级和三级。此外,检测到9个阳性选择位点(BsupanD中的A13、T14、V23、L32、V44、N49、L55、L78和V85)。如SDS-PAGE测定和对BsupanD和EcoPanD突变体的催化活性测定所示,其中三个位点(T14,V44和V85)影响PanD活性,并参与panD自切割的分歧,而其他6个位点仅影响PanD的酶活性。此外,结构分析表明,影响催化的9个位点的结构机理是不同的。总之,揭示了导致PanD自切割分化的三个位点,为PanD在β-丙氨酸合成中的工业应用奠定了基础。
L-aspartate-α-decarboxylase (PanD) is an essential enzyme catalysing the decarboxylation of L-aspartate to β-alanine in organisms. To perform the catalytic functions, PanD pro-proteins need to be self-cleaved to form two subunits: active α-subunit and β-subunit. However, the processes of self-cleavage have diverged in different organisms for unknown reasons. To reveal the possible divergence mechanisms, the molecular evolution, selection pressures and site-directed mutagenesis of the panD gene family were explored in this study. The evolution analysis revealed that the panD genes in bacteria have diverged into three clades: Class I, Class II and Class III. Furthermore, 9 positive selection sites (A13, T14, V23, L32, V44, N49, L55, L78, and V85 in BsupanD) were detected. As shown by SDS-PAGE assay and catalytic activity determination in the mutants of BsupanD and EcoPanD, three of those sites (T14, V44 and V85) affect the PanD activities and are involved in the divergence of panD self-cleavage, while the other 6 sites only influenced the enzymatic activities of PanD. Furthermore, the structure analysis indicated that the structural mechanisms of the 9 sites affecting the catalysis were various. In all, three sites contributing to the divergence of PanD self-cleavage were revealed, and the results also provide foundation for the industrial application of PanD in β-alanine synthesis.