The Three-dimensional Structures of Two β-Agarases

• Published in: The Journal of biological chemistry 2003-11, Vol.278 (47), p.47171-47180
• Main Authors: Allouch, Julie, Jam, Murielle, Helbert, William, Barbeyron, Tristan, Kloareg, Bernard, Henrissat, Bernard, Czjzek, Mirjam
• Format: Article
• Language: English
• Subjects: Bacteria; Bacterial Proteins - chemistry; Binding Sites; Catalytic Domain; Crystallization; Crystallography, X-Ray; Flavobacteriaceae - enzymology; Glycoside Hydrolases - chemistry; Marine; Protein Conformation; Rhodophyta; Zobellia galactanivorans
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M308313200

Agars are important gelifying agents for biochemical use and the food industry. To cleave the β-1,4-linkages between β-d-galactose and α-l-3,6-anhydro-galactose residues in the red algal galactans known as agars, marine bacteria produce polysaccharide hydrolases called β-agarases. β-Agarases A and B from Zobellia galactanivorans Dsij have recently been biochemically characterized. Here we report the first crystal structure of these two β-agarases. The two proteins were overproduced in Escherichia coli and crystallized, and the crystal structures were determined at 1.48 and 2.3 Å for β-agarases A and B, respectively. The structure of β-agarase A was solved by the multiple anomalous diffraction method, whereas β-agarase B was solved with molecular replacement using β-agarase A as model. Their structures adopt a jelly roll fold with a deep active site channel harboring the catalytic machinery, namely the nucleophilic residues Glu-147 and Glu-184 and the acid/base residues Glu-152 and Glu-189 for β-agarases A and B, respectively. The structures of the agarases were compared with those of two lichenases and of a κ-carrageenase, which all belong to family 16 of the glycoside hydrolases in order to pinpoint the residues responsible for their widely differing substrate specificity. The relationship between structure and enzymatic activity of the two β-agarases from Z. galactanivorans Dsij was studied by analysis of the degradation products starting with different oligosaccharides. The combination of the structural and biochemical results allowed the determination of the number of subsites present in the catalytic cleft of the β-agarases.