Enzymatic Hydrolysis of Human Milk Oligosaccharides. The Molecular Mechanism of Bifidobacterium Bifidum Lacto-N-biosidase

Bifidobacterium bifidumlacto-N-biosidase (LnbB) isacriticalenzymeforthedegradationofhumanmilkoligosaccharides in the gut microbiota of breast-fed infants.Guided by recent crystal structures, we unveil its molecularmechanism of catalysis using QM/MM metadynamics. We showthat the oligosaccharide substrate follows1S3/1,4B ->[4E]double dagger -> 4C1/4H5and4C1/4H5 ->[4E/4H5]double dagger -> 1,4Bconformationalitineraries for the two successive reaction steps, with reactionfree energy barriers in agreement with experiments. Thesimulations also identify a critical histidine (His263) that switchesbetween two orientations to modulate the pKaof the acid/baseresidue, facilitating catalysis. The reaction intermediate of LnbB isbest depicted as an oxazolinium ion, with a minor population of neutral oxazoline. The present study sheds light on the processing ofoligosaccharides of the early life microbiota and will be useful for the engineering of LnbB and similar glycosidases for biocatalysis.