Enhanced Synthesis Ability of a β-1,3-Galactosyltransferase from <i>Proteus mirabilis</i> with Mechanistic Dissection of Transglycosylation

Lacto-<i>N</i>-tetraose (LNT), a core component in human milk oligosaccharides (HMOs), has many excellent physiological effects. β-1,3-Galactosyltransferase (β-1,3-GalT)-mediated synthesis of LNT from UDP-galactose (UDP-Gal) and lacto-<i>N</i>-triose II (LNT II) is a promising approach. However, few β-1,3-GalTs suitable for LNT synthesis have been reported, and their unclear catalytic mechanism limits efficient LNT synthesis. Here, we identified a novel LNT-producing β-1,3-GalT from <i>Proteus mirabilis</i> (<i>Pm</i>β3GalT). The enzyme showed optimal activity at pH 7.5 and 50 °C and exhibited an 8.4-fold higher catalytic rate toward UDP-Gal than the previously reported EcWbgO. Furthermore, the mutant <i>Pm</i>β3GalT (S207H) displayed a 2.57-fold increase in transglycosylation activity over the wild type. We elucidated the enhanced stability and activity to internal conformational adjustments and the shifting of its flexible regions. This study not only provides a novel β-1,3-GalT for LNT synthesis but also elucidates the mechanism for enhancing transglycosylation activity, providing insights into the structure-function relationship of these enzymes.