Engineering Bifidobacterium longum Endo-α-N-acetylgalactosaminidase for Neu5Acα2-3Galβ1-3GalNAc reactivity on Fetuin

Carbohydrate-active enzyme (CAZyme) discovery and engineering via (Ultra)high-throughput screening

“…This is commonly carried out with detection systems such as the DNS assay, allowing use of natural carbohydrates or glycoproteins as substrates. [45][46][47] One of the great benefits of plate-based screening is that many different methods of detection and sample manipulation are possible. While too numerous to list all examples, we have highlighted some interesting versions based upon this basic concept throughout this review.…”

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.…”

“…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. 15 , 16 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. 17 − 19 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.…”