Enterococcus faecalis α1–2‐mannosidase (EfMan‐I): an efficient catalyst for glycoprotein N‐glycan modification

Abstract

While multiple α 1–2‐mannosidases are necessary for glycoprotein N‐glycan maturation in vertebrates, a single bacterial α1–2‐mannosidase can be sufficient to cleave all α1–2‐linked mannose residues in host glycoprotein N‐glycans. We report here the characterization and crystal structure of a new α1–2‐mannosidase (EfMan‐I) from Enterococcus faecalis, a Gram‐positive opportunistic human pathogen. EfMan‐I catalyzes the cleavage of α1–2‐mannose from not only oligomannoses but also high‐mannose‐type N‐glycans on glycoproteins. Its 2.15 Å resolution crystal structure reveals a two‐domain enzyme fold similar to other CAZy GH92 mannosidases. An unexpected potassium ion was observed bridging two domains near the active site. These findings support EfMan‐I as an effective catalyst for in vitro N‐glycan modification of glycoproteins with high‐mannose‐type N‐glycans.

Unlocking the Hydrolytic Mechanism of GH92 α‐1,2‐Mannosidases: Computation Inspires the use of C‐Glycosides as Michaelis Complex Mimics

“…Since no experimental evidence of the ring distortion existed, our calculation would have appeared just as a theoretical hypothesis. However, two recent works in organic chemistry and biochemistry inspired a strategy to demonstrate distortion of α‐1,2‐mannobiose 1 when forming a Michaelis complex with GH92 enzymes [24–26] …”

“…However, two recent works in organic chemistry and biochemistry inspired a strategy to demonstrate distortion of α‐1,2‐mannobiose 1 when forming a Michaelis complex with GH92 enzymes. [ 24 , 25 , 26 ]…”

Plant N-glycan breakdown by human gut Bacteroides

“…The eight GH92 structures already available include five α-1,2-mannosidases, an α-1,3mannosidase, an α-1,4-mannosidase, and a mannose-α-1,4-PO4-mannose mannosidase (15)(16)(17)(18)(19). Previous comparison of the α-1,2-mannosidase structures revealed three residues coordinating the mannose at the +1 subsite that drive specificity for α-1,2-linkages.…”

Unlocking the hydrolytic mechanism of GH92 α-1,2-mannosidases: computation inspires using C-glycosides as Michaelis complex mimics

“…However, two recent works in organic chemistry and biochemistry inspired a strategy to demonstrate distortion of α-1,2-mannobiose 1 when forming a Michaelis complex with GH92 enzymes. [23][24][25] On the one hand, the synthesis and conformational analysis of Coligosaccharides 22,23 provided an evidence of a group of molecules capable to mimic natural carbohydrates. On the other hand, Li et al were capable to characterize and crystallize a Ca 2+ -dependent α-1,2-mannosidase from Enterococcus faecalis (EfGH92), which has two domains like other GH92 mannosidases.…”

“…On the other hand, Li et al were capable to characterize and crystallize a Ca 2+ -dependent α-1,2-mannosidase from Enterococcus faecalis (EfGH92), which has two domains like other GH92 mannosidases. 25 Structural superposition between BtGH92 and EfGH92 is provided in the SI.…”