Asp271 is critical for substrate interaction with the surface binding site in β‐agarase a from Zobellia galactanivorans

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Casper Wilkens, Manish Kumar Tiwari, Helen Webb-Thomasen, Murielle Jam, Mirjam Czjzek, Birte Svensson

In the marine environment agar degradation is assured by bacteria that contain large agarolytic systems with enzymes acting in various endo- and exo-modes. Agarase A (AgaA) is an endo-glycoside hydrolase of family 16 considered to initiate degradation of agarose. Agaro-oligosaccharide binding at a unique surface binding site (SBS) in AgaA from Zobellia galactanivorans was investigated by computational methods in conjunction with a structure/sequence guided approach of site-directed mutagenesis probed by surface plasmon resonance binding analysis of agaro-oligosaccharides of DP 4-10. The crystal structure has shown that agaro-octaose interacts via H-bonds and aromatic stacking along 7 subsites (L through R) of the SBS in the inactive catalytic nucleophile mutant AgaA-E147S. D271 is centrally located in the extended SBS where it forms H-bonds to galactose and 3,6-anhydrogalactose residues of agaro-octaose at subsites O and P. We propose D271 is a key residue in ligand binding to the SBS. Thus AgaA-E147S/D271A gave slightly decreasing K D values from 625 ± 118 to 468 ± 13 μM for agaro-hexaose, -octaose, and -decaose, which represent 3- to 4-fold reduced affinity compared with AgaA-E147S. Molecular dynamics simulations and interaction analyses of AgaA-E147S/D271A indicated disruption of an extended H-bond network supporting that D271 is critical for the functional SBS. Notably, neither AgaA-E147S/W87A nor AgaA-E147S/W277A, designed to eliminate stacking with galactose residues at subsites O and Q, respectively, were produced in soluble form. W87 and W277 may thus control correct folding and structural integrity of AgaA.

Original languageEnglish
JournalProteins - Structure Function and Bioinformatics
Issue number1
Pages (from-to)34-40
Number of pages7
Publication statusPublished - 2019
Externally publishedYes

    Research areas

  • agaro-oligosaccharides, comparative modeling, glycoside hydrolase family 16, marine bacterium, mutational analysis, surface binding site, surface plasmon resonance, β-agarase

ID: 209113124