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

Abstract

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 p K 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.

Role of Enzyme and Active Site Conformational Dynamics in the Catalysis by α-Amylase Explored with QM/MM Molecular Dynamics

“…A set of 70 atoms was defined to undergo QM calculations at the PBE/def2-SVP level (Figure , right): the methyl–carboxylic terminus of acidic Glu233, nucleophilic Asp197, and transition state stabilizer Asp300; the glucose monomers involved in the scissile glycosidic bond of the substrate; and a water molecule near the acidic oxygen of Glu233 and the oxygen of the scissile glycosidic bond (previously shown to be important for catalysis). The combination of PBE and small basis sets has been widely used to study reaction mechanisms of glycosidases, − as well as to accurately describe the conformational diversity of several carbohydrate units (including the glucopyranose ring composing the 5-GA substrate), , and it was also observed to accurately reproduce geometries for the glycosylation reaction with moderately low computational effort . The use of single split-valence polarization basis sets was also indicated to reduce overpolarization effects in QM/MM MD simulations .…”

Substrate-Assisted Mechanism for the Degradation of N-Glycans by a Gut Bacterial Mannoside Phosphorylase

“…DFT was used to describe the atoms of the QM region, along with the PBE functional and a plane wave basis set (70 Ry cutoff), whereas the atoms of the MM region (195513 atoms) were treated with the AMBER force field . This approach has previously been used to model mechanisms in carbohydrate-active enzymes, − including the phosphorolysis reaction in an engineered GH …”

Mechanistic and Structural Insights into the Specificity and Biological Functions of Bacterial Sulfoglycosidases

“…The direct W409–E391 interaction seen in the Bt4394 D335N -6S-NAG-oxazoline intermediate structure delivers the important closure of the essential catalytic loop through the formation of a direct H-bond from the E336 backbone amide to E391, which is not the case in the BbhII E553Q -6S-NAG-oxazoline intermediate structure. Moreover, the conserved catalytically important histidine (H467 from BbhII and H270 from Bt4394) has been proposed, quantum mechanically, to modulate the p K a s of the catalytic Glu–Asp diad by oscillating between them in a GH20 family endo Lacto- N -biosidase LnbB . The unaltered position of histidine with different catalytic loop conformations observed in our two 6S-oxazoline intermediate structures has provided another possibility for this histidine to achieve the same interaction and modulation of the catalytic diad by dynamic movements of the catalytic loop (Figure c,d).…”

“…Moreover, the conserved catalytically important histidine (H467 from BbhII and H270 from Bt4394) has been proposed, quantum mechanically, to modulate the pK a s of the catalytic Glu−Asp diad by oscillating between them in a GH20 family endo Lacto-N-biosidase LnbB. 63 The unaltered position of histidine with different catalytic loop conformations observed in our two 6S-oxazoline intermediate structures has provided another possibility for this histidine to achieve the same interaction and modulation of the catalytic diad by dynamic movements of the catalytic loop (Figure 2c,d). This is further exemplified by the 3000-fold difference in the activities of His-to-Phe variants relative to the wild-type enzymes.…”