期刊
ENZYME AND MICROBIAL TECHNOLOGY
卷 158, 期 -, 页码 -出版社
ELSEVIER SCIENCE INC
DOI: 10.1016/j.enzmictec.2022.110048
关键词
L-aspartate-alpha-decarboxylase; Self-cleavage; Bacillus subtilis; beta-Alanine; Site-directed mutagenesis
资金
- Natural Science Foundation of Zhejiang Province, People's Republic of China - Science Technology Department of Zhejiang Province, People's Republic of China [LY21H280009]
This study investigates the molecular evolution, selection pressures, and site-directed mutagenesis of the panD gene family. It reveals that panD genes in bacteria have diverged into three clades and identifies three sites that affect the catalytic activity and self-cleavage divergence of PanD.
L-aspartate-alpha-decarboxylase (PanD) is an essential enzyme catalysing the decarboxylation of L-aspartate to beta-alanine in organisms. To perform the catalytic functions, PanD pro-proteins need to be self-cleaved to form two subunits: active alpha-subunit and beta-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 beta-alanine synthesis.
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