<div>Retaining glycoside hydrolases cleave their substrates through stereochemical retention at the anomeric position. Typically, this involves two-step mechanisms using either an enzymatic nucleophile via a covalent glycosyl enzyme intermediate, or neighboring group participation by a substrate-borne 2-acetamido neighboring group via an oxazoline intermediate; no enzymatic mechanism with participation of the sugar 2-hydroxyl has been reported. Here, we detail structural, computational and kinetic evidence for neighboring group participation by a mannose 2-hydroxyl in glycoside hydrolase family 99 endo-α-1,2-mannanases. We present a series of crystallographic snapshots of key species along the reaction coordinate: a Michaelis complex with a tetrasaccharide substrate; complexes with intermediate mimics, β-1,2-aziridine and β-1,2-epoxide; and a product complex. The 1,2-epoxide intermediate mimic displayed hydrolytic and transfer reactivity analogous to that expected for the 1,2-anhydro sugar intermediate supporting its catalytic equivalence. Quantum mechanics/molecular mechanics modelling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar via a transition state in an unprecedented flattened, envelope (<i>E</i>₃) conformation. Kinetic isotope effects for anomeric-²H and anomeric-¹³C support an oxocarbenium ion-like transition state and that for C2-¹⁸O (1.052 ± 0.006) directly implicates nucleophilic participation by the C2-hydroxyl. Collectively, these data substantiate this unprecedented and long-imagined enzymatic mechanism.</div><div><br></div>

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Rational feeding from the early days of life programs the health, growth and development of the child over the following years. By the age of 6 months children on artificial feeding are reliably more likely to form excess body weight. The nature of nutrition depends on the condition of the overall and local immune protection of the child. Application of non-invasive methods for determining sIgA (in oral fluid (saliva), in coprofiltrate) allows to assess local immunity in infancy age. Breastfed infants receive sIgA with mother’s milk and have additional protection against the pathogens of respiratory and intestinal infections. The level of sIgA in the mother’s breast milk can be determined to assess the condition of children’s immunity. Children on breastfeeding experience acute respiratory diseases less often compared to children fed with adapted milk formula.

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