Figure 4. Representative amino acids transformations of
variants W1-W3 and binding pose analysis in the docking model.(A)
Representative amino acids transformation results of variants W1-W3.
(B-G) Posture analysis of variant W1 or W2 after docking with
representative substrates (L-Leu, L-Met, l-val, L-Phe, L-Arg or L-Glu).
We also evaluated the properties of variant W2 for different amino
acids. As shown in Supplementary Table 3, variant W2 significantly
increased catalytic efficiency toward L-Phe and L-Met, but not toward
the other substrates. D1Phe decreased the most (15.4%;
from 3.0 to 2.6 Å), which increased its kcat ,Km ,kcat/Km , and specific activity
toward L-Phe by 107.1%, 23.5%, 64.7%, and 65.5%, respectively.
D1Met decreased by 14.7% (from 2.8 to 2.4 Å), which
increased kcat , Km ,kcat/Km , and specific activity by
57.7%, 26.8%, 23.8%, and 38.2%, respectively.
The mechanisms underlying improved catalytic efficiency of variant W2
were analyzed. Compared to Pmi LAAD, molecular dynamic simulations
revealed an increase in root-mean-square fluctuations (RMSFs) for six
residues (T105, D144, E145, E340, S412, and E417) around the substrate
channel (Figure 5). The increased conformational dynamic of these
mutated residues resulted in greater structural flexibility of the
channel(W. Song et al. 2020; Yang et al. 2017), which
changed the orientation of the substrate side-chain (phenyl or
methylthio group) (Figure 4C and 4E). As a consequence, the binding
posture of L-Phe and L-Met was significantly altered, decreasing θ of
W2Phe and W2Met from 136.3° to 94.8° and
from 128.2° to 96.8°, respectively. The reduced θ shortened
D1Phe and D1Met by 0.4 Å, resulting in
higher catalytic efficiency toward L-Phe and L-Met. Meanwhile, θ and D1
in W2 remained almost unchanged toward L-Leu, L-Val, L-Arg, and L-Glu
(Figure 4B–4G), and so was their catalytic efficiency.