The agar-specific hydrolase Zg AgaC from the marine bacterium Zobellia galactanivorans defines a new GH16 protein subfamily

J Biol Chem. 2019 Apr 26;294(17):6923-6939. doi: 10.1074/jbc.RA118.006609. Epub 2019 Mar 7.


Agars are sulfated galactans from red macroalgae and are composed of a d-galactose (G unit) and l-galactose (L unit) alternatively linked by α-1,3 and β-1,4 glycosidic bonds. These polysaccharides display high complexity, with numerous modifications of their backbone (e.g. presence of a 3,6-anhydro-bridge (LA unit) and sulfations and methylation). Currently, bacterial polysaccharidases that hydrolyze agars (β-agarases and β-porphyranases) have been characterized on simple agarose and more rarely on porphyran, a polymer containing both agarobiose (G-LA) and porphyranobiose (GL6S) motifs. How bacteria can degrade complex agars remains therefore an open question. Here, we studied an enzyme from the marine bacterium Zobellia galactanivorans (ZgAgaC) that is distantly related to the glycoside hydrolase 16 (GH16) family β-agarases and β-porphyranases. Using a large red algae collection, we demonstrate that ZgAgaC hydrolyzes not only agarose but also complex agars from Ceramiales species. Using tandem MS analysis, we elucidated the structure of a purified hexasaccharide product, L6S-G-LA2Me-G(2Pentose)-LA2S-G, released by the activity of ZgAgaC on agar extracted from Osmundea pinnatifida By resolving the crystal structure of ZgAgaC at high resolution (1.3 Å) and comparison with the structures of ZgAgaB and ZgPorA in complex with their respective substrates, we determined that ZgAgaC recognizes agarose via a mechanism different from that of classical β-agarases. Moreover, we identified conserved residues involved in the binding of complex oligoagars and demonstrate a probable influence of the acidic polysaccharide's pH microenvironment on hydrolase activity. Finally, a phylogenetic analysis supported the notion that ZgAgaC homologs define a new GH16 subfamily distinct from β-porphyranases and classical β-agarases.

Keywords: GH16; agar; algae; bacteria; crystal structure; evolution; glycoside hydrolase; marine bacteria; mass spectrometry; pH microenvironment; polysaccharide; red algae; sulfated polysaccharide.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Agar / metabolism*
  • Amino Acid Sequence
  • Aquatic Organisms / enzymology
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / isolation & purification*
  • Bacterial Proteins / metabolism
  • Crystallography, X-Ray
  • Flavobacteriaceae / enzymology*
  • Hydrogen-Ion Concentration
  • Hydrolases / chemistry
  • Hydrolases / isolation & purification*
  • Hydrolases / metabolism
  • Phylogeny
  • Protein Conformation
  • Seawater / microbiology


  • Bacterial Proteins
  • Agar
  • Hydrolases

Supplementary concepts

  • Zobellia galactanivorans

Associated data

  • PDB/1O4Y
  • PDB/1O4Z
  • PDB/3ILF
  • PDB/3JUU
  • PDB/4ATE
  • PDB/2YCB
  • PDB/5FD3
  • PDB/4ATF
  • PDB/5NBO
  • PDB/3WVJ
  • PDB/5WUT
  • PDB/6IBW
  • PDB/5JVV
  • PDB/3WDT
  • PDB/2CL2
  • PDB/1UMZ
  • PDB/2UWA
  • PDB/5DZE
  • PDB/6HY3