Protein engineering to increase the catalytic efficiency ofPmiLAAD
The substrate and FAD binding domains were considered to be the key factors affecting D1. Nine potential amino acid residues in these two regions (S98, D149, K150, P203, V276, L278, S279, H295, and H301) were selected and subjected to alanine scanning (Supplementary Figure 1). S98A and D149A emerged as the ones causing the greatest increase in relative activity toward Val, Arg, and Glu: 13.6%, 22.3%, and 34.2% (S98A), and 10.1%, 14.3%, and 30.0% (D149A), respectively (Supplementary Figure 2A). Saturation of mutagenesis yielded the single variants S98A and D149Y, whose relative activity toward Val, Arg, and Glu had increased by 23.2%, 22.6%, and 21.4%, respectively (Supplementary Table 2). Then, these two variants were combined to obtain Pmi LAADS98A/D149Y (W1) (Supplementary Figure 3), which significantly improved the catalytic efficiency toward short-chain aliphatic amino acids (L-Val, 85.8%) or charged amino acids (L-Arg, 65.1%; L-Glu, 51.7%), but not toward large-volume aromatic L-Phe or sulfur-containing L-Met (Table 1). Docking simulation results revealed a stronger hydrophobic interaction and van der Waals forces between the substrate channel and the side chain of L-Phe or L-Met, which likely affected D1. Therefore, ten dynamic unstable amino acid residues, i.e., the residues whose RMSFs values fluctuates greatly (T105, I109, F110, D144, E145, K146, F317, E340, S412, and E417) near the substrate channel were selected through conformational kinetics (Supplementary Figure 4) and subjected to alanine scanning. T105A, D144A, E145A, E340A, S412A, and E417A emerged as the ones increasing the relative activity toward Phe and Met by ≥10% (Supplementary Figure 2B). Finally, the six variants were combined to generate Pmi LAADT105A/D144A/E145A/E340A/S412A/E417A (W2) (Supplementary Figure 5), which exhibited higher catalytic efficiency toward bulkier substrates, including L-Phe (165.9%), L-Trp (151.5%), L-Tyr (155.8%), and L-Met (123.9%), compared to Pmi LAAD (Table 1).
Table 1. Comparison table of transformation of strain S3 with WT, variants W1 and W2.