Identification of mutations restricting autocatalytic activation of bacterial L-aspartate α-decarboxylase.

Bacterial L-aspartate α-decarboxylase (PanD) specifically catalyzes the decarboxylation of L-aspartic acid to β-alanine. It is translated as an inactive pro-protein, then processed by self-cleavage to form two small subunits with catalytic activity. There is a significant difference in the efficiency of this process among the reported PanDs, while the structural basis remains unclear. More PanDs with known sequences and characterized properties are needed to shed light on the molecular basis of the self-cleavage process. In this study, PanD genes from 33 selected origins were synthesized and expressed; using purified recombinant enzymes, their self-processing properties were characterized and classified. Three classes of PanDs were acquired based on their self-cleavage efficiency. Combined with the phylogenetic analysis and structure comparison, sited-directed mutagenesis was performed to investigate the effects of four mutants on self-processing. In comparison with the wild-type (96.4%), the self-cleavage efficiencies of mutants V23E, I26C, T27A, and E56S were decreased to 90.5, 83.6, 74.4 and 81.2%, respectively. The results indicated that residues of V23, I26, T27 and E56 were critical to the self-cleavage processing of PanDs. This work provided further understanding to the self-cleavage processing of PanDs, which may contribute to protein engineering of the enzyme.