Enhancing thermostability of lysine hydroxylase via a semi-rational design

(2S,4 R)-4-Hydroxylysine (4-OH-Lys), a derivative of L-lysine, possesses a unique chemical structure that makes it a crucial precursor for the synthesis of pharmaceutical molecules, with extensive applications in the pharmaceutical and biochemical industries. Lysine hydroxylase (K4H) catalyzes the conversion of L-lysine to 4-OH-Lys, offering advantages such as mild reaction conditions, straightforward reaction steps, good regioselectivity, and high catalytic efficiency compared to chemical synthesis and natural extraction methods. However, the low thermostability of K4H hinders its application in large-scale production. In this study, we employed a semi-rational design approach, guided by ΔΔG folding free energy calculations and message-passing neural networks to enhance the thermostability of K4H. After two rounds of evolution, we identified two beneficial mutants: M25 (S101P/Q257M) and M32 (Q257M/V298I). Thermostability assessments revealed that the half-lives (t_1/2) of M25 and M32 at 40 °C were 23.9-fold and 13.3-fold higher than that of the wild-type (WT), with melting temperatures (T_m) exceeding those of WT by 4.2 °C and 8.3 °C, respectively. Molecular dynamics simulations illuminated the mechanisms underlying this enhanced thermostability. This work provides valuable insights into the thermostability of K4H and yields key mutants that are promising candidates for practical production of 4-OH-Lys.