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

Bifidobacterium bifidum lacto-N-biosidase (LnbB) is a critical enzyme for the degradation of human milk oligosaccharides in the gut microbiota of breast-fed infants. Guided by recent crystal structures, we unveil its molecular mechanism of catalysis using QM/MM metadynamics. We show that the oligosaccharide substrate follows 1 S 3/1,4 B → [4 E]‡ → 4 C 1/4 H 5 and 4 C 1/4 H 5 → [4 E/4 H 5]‡ → 1,4 B conformational itineraries for the two successive reaction steps, with reaction free energy barriers in agreement with experiments. The simulations also identify a critical histidine (His263) that switches between two orientations to modulate the pK a of the acid/base residue, facilitating catalysis. The reaction intermediate of LnbB is best depicted as an oxazolinium ion, with a minor population of neutral oxazoline. The present study sheds light on the processing of oligosaccharides of the early life microbiota and will be useful for the engineering of LnbB and similar glycosidases for biocatalysis.

Computer Simulation to Rationalize "Rational" Engineering of Glycoside Hydrolases and Glycosyltransferases.

Glycoside hydrolases and glycosyltransferases are the main classes of enzymes that synthesize and degrade carbohydrates, molecules essential to life that are a challenge for classical chemistry. As such, considerable efforts have been made to engineer these enzymes and make them pliable to human needs, ranging from directed evolution to rational design, including mechanism engineering. Such endeavors fall short and are unreported in numerous cases, while even success is a necessary but not sufficient proof that the chemical rationale behind the design is correct. Here we review some of the recent work in CAZyme mechanism engineering, showing that computational simulations are instrumental to rationalize experimental data, providing mechanistic insight into how native and engineered CAZymes catalyze chemical reactions. We illustrate this with two recent studies in which (i) a glycoside hydrolase is converted into a glycoside phosphorylase and (ii) substrate specificity of a glycosyltransferase is engineered toward forming O-, N-, or S-glycosidic bonds.