Structural and functional characterization of PL28 family ulvan lyase NLR48 from Nonlabens ulvanivorans

J Biol Chem. 2018 Jul 20;293(29):11564-11573. doi: 10.1074/jbc.RA118.003659. Epub 2018 Jun 6.

Abstract

Ulvan is a complex sulfated polysaccharide present in the cell wall of green algae of the genus Ulva (Chlorophyta). The first ulvan-degrading polysaccharide lyases were identified several years ago, and more were discovered through genome sequencing of marine bacteria. Ulvan lyases are now grouped in three polysaccharide lyase (PL) families in the CAZy database, PL24, PL25, and PL28. The recently determined structures of the representative lyases from families PL24 and PL25 show that they adopt a seven-bladed β-propeller fold and utilize the His/Tyr catalytic mechanism. No structural information is yet available for PL28 ulvan lyases. NLR48 from Nonlabens ulvanivorans belongs to PL28 together with its close paralog, NLR42. Biochemical studies of NLR42 have revealed that it can cleave ulvan next to both uronic acid epimers. We report the crystal structure of ulvan lyase NLR48 at 1.9-Å resolution. It has a β-jelly roll fold with an extended, deep, and positively charged substrate-binding cleft. Putative active-site residues were identified from the sequence conservation pattern, and their role was confirmed by site-directed mutagenesis. The structure of an inactive K162M mutant with a tetrasaccharide substrate showed the substrate occupying the "-" subsites. Comparison with lyases from other PL families with β-jelly roll folds supported assignment of the active site and explained its ability to degrade ulvan next to either epimer of uronic acid. NLR48 contains the His/Tyr catalytic machinery with Lys162 and Tyr281 playing the catalytic base/acid roles.

Keywords: PL28 family; algae; bacteria; carbohydrate processing; catalysis; crystal structure; substrate binding; substrate specificity; ulvan lyase; β-jelly roll fold.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Catalytic Domain
  • Crystallography, X-Ray
  • Flavobacteriaceae / chemistry
  • Flavobacteriaceae / enzymology*
  • Flavobacteriaceae / metabolism
  • Models, Molecular
  • Oligosaccharides / metabolism
  • Polysaccharide-Lyases / chemistry
  • Polysaccharide-Lyases / metabolism*
  • Protein Conformation
  • Substrate Specificity

Substances

  • Oligosaccharides
  • Polysaccharide-Lyases
  • ulvan-lyase

Associated data

  • PDB/2ZAA
  • PDB/3INA
  • PDB/4Q8K
  • PDB/2ZZJ
  • PDB/3A0N
  • PDB/2INA
  • PDB/6D2C
  • PDB/6D3U

Grant support