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. 2022 Apr 15;12(8):4737-4743.
doi: 10.1021/acscatal.2c00309. Epub 2022 Apr 6.

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

Irene Cuxart  1 , Joan Coines  1 , Oriol Esquivias  1 , Magda Faijes  2 , Antoni Planas  2 , Xevi Biarnés  2 , Carme Rovira  1   3
Affiliations

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

Irene Cuxart et al. ACS Catal. .

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 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.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
(A) Hydrolysis of the human milk oligosaccharise lacto-N-tetraose (LNT) catalyzed by Bifidobacterium bifidum lacto-N-biosidase (LnbB). (B) Substrate-assisted reaction catalyzed by LnbB.
Figure 2
Figure 2
Structure of LnbB in complex with lacto-N-tetraose (LNT) obtained from MD and QM/MM MD simulations.
Figure 3
Figure 3
First step of the chemical reaction catalyzed by LnbB. (A) Reaction free energy surface as a function of depicted CVs. (B) Most relevant states along the reaction coordinate. (C) Evolution of relevant distances along the reaction coordinate.
Figure 4
Figure 4
Analysis of the dynamics of water molecules in the active site at the reaction intermediate. Multiple replicas of the simulation (total 300 ns) gave similar results.
Figure 5
Figure 5
Second step of the chemical reaction catalyzed by LnbB. (A) Collective variables used to drive the metadynamics bias and reconstructed reaction free energy surface. (B) Main states along the reaction coordinate. (C) Evolution of relevant distances along the reaction coordinate.
See this image and copyright information in PMC

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