Identification of a Catalytic Nucleophile-Activating Network in the endo-α-N-Acetylgalactosaminidase of Family GH101

Abstract

Bifidobacterium longum endo-α-N-acetylgalactosaminidase (GH101), EngBF, is highly specific toward the mucin Core 1 glycan, Galβ1-3GalNAc. Apart from the side chains involved in the retaining mechanism of EngBF, Asp-682 is important for the activity. In the crystal structures of both EngBF and EngSP (from Streptococcus pneumoniae), we identified a conserved water molecule in proximity to Asp-682 and the homologue residue in EngSP. The water molecule also coordinates the catalytic nucleophile and three other residues conserved in GH101 enzymes; in EngBF, these residues are His-685, His-718, and Asn-720. With casein-glycomacropeptide as the substrate, the importance of Asp-682 was confirmed by the lack of a detectable activity for the D682N enzyme. The enzyme variants, H685A, H718A, H685Q, and H718Q, all displayed only a modestly reduction in k cat of up to 15 fold for the H718A variant. However, the double-substituted variants, H685A/H718A and H685Q/H718Q, had a greatly reduced k cat value by about 200 fold compared to that of wild-type EngBF. With the synthetic substrate, Galβ(1−3)GalNAcα1-paranitrophenol, k cat of the double-substituted variants was only up to 30-fold reduced and was found to increase with pH. Compared to the pre-steady-state kinetics of wild-type EngBF, a burst of about the size of the enzyme concentration was absent with the doublesubstituted EngBF variants, indicating that the nucleophilic attack had become at least as slow as the hydrolysis of the enzyme intermediate. Together, the results indicate that not only Asp-682 but also the entire conserved network of His-685, His-718, and what we suggest is a catalytic water molecule is important in the activation of the catalytic nucleophile.

Carving out a Glycoside Hydrolase Active Site for Incorporation into a New Protein Scaffold Using Deep Network Hallucination

“…The endo-α- N -acetylgalactosaminidases (EC 3.2.1.97) of the GH101 enzyme family as classified in the CAZy database () catalyzes the hydrolysis of the mucin O -glycan, Galβ(1–3)­GalNAc. , Despite the more than 1350 amino acid residues large multidomain architecture of the GH101 family, the domain constituting the actual catalytic site is composed of only approximately 300 amino acids, forming a distorted (β/α) 8 TIM-barrel-like fold sharing structural similarity with the GH13 α-amylase family. − The substrate binding pocket of endo-α- N -acetylgalactosaminidases from Streptococcus pneumoniae (EngSP) and Bifidobacterium longum (EngBF) is shaped to complement the Galβ(1–3)­GalNac substrate, whereas specific hydrogen bonds and local conformational changes involving a conserved tryptophan “lid” contribute to an occluded bound state. , The anomeric carbon of Galβ(1–3)­GalNac is positioned in close proximity to the catalytic nucleophile and acid/base that are central to the double-displacement mechanism retaining the stereochemistry of the glycan. − The remaining structural domains presumably play crucial roles in maintaining overall enzyme stability and solubility or in providing functions such as macromolecular substrate recognition. Creating a molecule with a simplified scaffold and reduced molecular size would provide a more manageable GH101 template enzyme for engineering efforts aimed at processing and modifying complex glycans.…”

“… 17 , 18 The anomeric carbon of Galβ(1–3)GalNac is positioned in close proximity to the catalytic nucleophile and acid/base that are central to the double-displacement mechanism retaining the stereochemistry of the glycan. 20 − 22 The remaining structural domains presumably play crucial roles in maintaining overall enzyme stability and solubility or in providing functions such as macromolecular substrate recognition. Creating a molecule with a simplified scaffold and reduced molecular size would provide a more manageable GH101 template enzyme for engineering efforts aimed at processing and modifying complex glycans.…”