Analysis of the structure and substrate scope of chitooligosaccharide oxidase reveals high affinity for C2-modified glucosamines

FEBS Lett. 2020 Sep;594(17):2819-2828. doi: 10.1002/1873-3468.13854. Epub 2020 Jun 18.

Abstract

Chitooligosaccharide oxidase (ChitO) is a fungal carbohydrate oxidase containing a bicovalently bound FAD cofactor. The enzyme is known to catalyse the oxidation of chitooligosaccharides, oligomers of N-acetylated glucosamines derived from chitin degradation. In this study, the unique substrate acceptance was explored by testing a range of N-acetyl-d-glucosamine derivatives, revealing that ChitO preferentially accepts carbohydrates with a hydrophobic group attached to C2. The enzyme also accepts streptozotocin, a natural product used to treat tumours. Elucidation of the crystal structure provides an explanation for the high affinity towards C2-decorated glucosamines: the active site has a secondary binding pocket that accommodates groups attached at C2. Docking simulations are fully in line with the observed substrate preference. This work expands the knowledge on this versatile enzyme.

Keywords: chitooligosaccharides; covalent flavin; crystal structure; glucosamine; oxidation.

MeSH terms

  • Binding Sites
  • Chitin / analogs & derivatives*
  • Chitin / chemistry*
  • Chitin / metabolism
  • Chitosan
  • Cloning, Molecular
  • Crystallography, X-Ray
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Flavin-Adenine Dinucleotide / chemistry*
  • Flavin-Adenine Dinucleotide / metabolism
  • Fungal Proteins / chemistry*
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism
  • Fusarium / chemistry*
  • Fusarium / enzymology
  • Gene Expression
  • Genetic Vectors / chemistry
  • Genetic Vectors / metabolism
  • Glucosamine / analogs & derivatives
  • Glucosamine / metabolism
  • Kinetics
  • Models, Molecular
  • Oligosaccharides
  • Oxidoreductases / chemistry*
  • Oxidoreductases / genetics*
  • Oxidoreductases / metabolism
  • Protein Binding
  • Protein Conformation, alpha-Helical
  • Protein Conformation, beta-Strand
  • Protein Interaction Domains and Motifs
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Substrate Specificity
  • Thermodynamics

Substances

  • Fungal Proteins
  • Oligosaccharides
  • Recombinant Proteins
  • oligochitosan
  • Chitin
  • Flavin-Adenine Dinucleotide
  • Chitosan
  • Oxidoreductases
  • Glucosamine

Supplementary concepts

  • Fusarium graminearum