Evaluation of W1 and W2 performance and analysis of the enhancement mechanism
The catalytic properties of variant W1 toward different amino acids were evaluated. As illustrated in Supplementary Table 4, W1 significantly increased catalytic efficiency toward L-Val, L-Arg, and L-Glu; while only slightly improving catalysis of the other substrates. D1 toward Val decreased the most (24.1%; from 2.9 to 2.4 Å), resulting inkcat , Km ,kcat/Km , and specific activity increasing by 71.4%, 31.2%, 29.3%, and 86.1%, respectively. Shortening of D1 toward Arg (9.1%) and Glu (5.3%) led to an increase in kcat (53.8% and 52.5%),Km (28.5% and 33.4%),kcat/Km (25.1% and 23.2%), and specific activity (130.3% and 584.8%).
To identify the mechanisms underlying improved catalytic efficiency, the interaction force, substrate binding pose, and angle were compared. When S98 and D149 (hydrophobicity, -0.8 and -3.5) were mutated to A98 and Y149 (hydrophobicity, +1.8 and -1.3), the pocket hydrophobicity was increased. This change altered the hydrophobic interactions between S98 and substrate-COOH, and between Y149-OHand substrate-NH2 (Figure 3), α-hydrogen was improved to be more perpendicular against the plain of the isoalloxazine ring causing the angle θ between the substrates’ αC-H, FAD N(5), and FAD N(10) to approach 90°. The shortest D1 occurs when the substrate’s αC-H is perpendicular to the FAD isoalloxazine ring. Interestingly, we found that θ of W1Val decreased from 131.2° to 91.2°, W1Arg from 142.2° to 93.4°, and W1Glufrom 148.2° to 98.2°, which resulted in D1Val, D1Arg, and D1Glu to be reduced by 0.5, 0.3, and 0.2 Å, respectively (Figure 4). The shorter D1 in W1 led to an increase in catalytic efficiency toward L-Val, L-Arg, and L-Glu. Notably, θ and D1 remained almost unchanged with respect to L-Leu, L-Phe, and L-Met, possibly explaining why the catalytic efficiency of W1 toward these amino acids had not improved.