One-site polarity switch enhances catalytic efficiency and stability of D-allulose 3-epimerase via...

The structural foundation of enzyme efficiency frequently resides in the subtle equilibrium between localized flexibility and overarching rigidity. This study presents evidence that a singular amino acid alteration (E35H) in Arthrobacter psychrolactophilus D-allulose 3-epimerase (ApDAEase) enhances catalytic efficiency by 1.4 times relative to the wild-type, attributed to a 1.3-fold reduction in K_m and a 1.1-fold elevation in k_cat. Structural insights were obtained from models predicted by AlphaFold in conjunction with molecular dynamics simulations. These findings indicated that the E35H substitution diminished the local hydrogen-bond network at residue 35, reducing interactions from six to five, which consequently loosened constraints within the active site. Analysis of RMSF revealed that localized flexibility was heightened at residues 35 and 6, whereas global fluctuations exhibited a decrease, aligning with the stabilization of the scaffold. Furthermore, a newly proposed interaction between E152 and substrate O1 offered an extra anchoring effect. CD spectroscopy and thermal unfolding analyses revealed a slight enhancement in thermostability, with a melting temperature of 94.3 °C compared to 92.6 °C observed in the wild-type. Collectively, these findings highlight residue 35 as a critical functional hotspot, where the modulation of polarity effectively reestablishes a balance between flexibility and rigidity, thereby providing valuable predictive insights into the mechanism of improved catalysis in DAEase.