Figure 2 | Scientific Reports

Figure 2

From: Membrane binding of the insertion sequence of Proteus vulgaris L-amino acid deaminase stabilizes protein structure and increases catalytic activity

Figure 2

The INS-membrane interactions enhance the enzymatic activities and modulate the substrate preference of pvLAAD. (a) The cartoon drawing of the INS (blue) of pvLAAD. Seven of the conserved surface-exposed hydrophobic residues are drawn as sticks (magenta). (b) Membrane binding of INS-mutated pvLAADs were detected by membrane co-pelleting assays. pvLAADs co-pelleting with membrane or staying in supernatant were analyzed with SDS-PAGE. FL-pvLAAD was used as the positive control, and the double-truncated ΔN-ΔINS-pvLAAD was used as the negative control. Although the N-terminal Tat peptide is the major membrane-binding site of pvLAAD, a small fraction of ΔN-pvLAAD still co-pelleted with the liposome through the INS-mediated membrane binding. Two double-mutated ΔN-pvLAAD proteins showed the similar membrane-binding capability to the wild-type ΔN-pvLAAD. But, co-pelleting of the quintuple-mutant ΔN-pvLAAD (5M-ΔN-pvLAAD) could not be observed, and all of the 5M-ΔN-pvLAAD protein stayed in the supernatant, indicating that the quintuple mutation completely abolished the membrane-binding capability of the INS. (c) Catalytic activities of FL-pvLAAD and 5M-FL-pvLAAD with or without membrane presence were tested at three different time points (1, 10 and 60 min). FL-pvLAAD and 5M-FL-pvLAAD gave the similar activity when in the solution, but activity enhancement by membrane binding could be observed only for wild-type FL-pvLAAD but not for 5M-FL-pvLAAD. (d,e) Catalytic activities of wild-type FL-pvLAAD and 5M-FL-pvLAAD against twenty proteinogenic L-amino acids were tested by measuring the α-keto acid production using chromogenic reaction with 2,4-dinitrophenylhydrazine (DNP). Membrane binding enhanced the activities of FL-pvLAAD for most of the twenty proteinogenic L-amino acids, but with different folds. The highest activities were observed for Asn, Asp, His, Leu, Met, Phe, Trp and Tyr when membrane was added, which are significantly different to the favorable substrates of pvLAAD without membrane. In contrast, no activity enhancement and substrate spectrum shift were observed for the 5M-FL-pvLAAD.

Back to article page