Agars are important gelifying agents for biochemical use and the food industry. To cleave the ␤-1,4-linkages between ␤-D-galactose and ␣-L-3,6-anhydro-galactose residues in the red algal galactans known as agars, marine bacteria produce polysaccharide hydrolases called ␤-agarases. ␤-Agarases A and B from Zobellia galactanivorans Dsij have recently been biochemically characterized. Here we report the first crystal structure of these two ␤-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 ␤-agarases A and B, respectively. The structure of ␤-agarase A was solved by the multiple anomalous diffraction method, whereas ␤-agarase B was solved with molecular replacement using ␤-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 ␤-agarases A and B, respectively. The structures of the agarases were compared with those of two lichenases and of a -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 ␤-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 ␤-agarases.Agarose is a hydrophilic polysaccharide found in the cell wall of marine red algae (Rhodophyceaea) (1), where it naturally occurs in the form of a pseudocrystalline matrix associated with cellulose (2). It consists of a linear backbone of galactopyranose residues linked by alternating ␣-1,3-and ␤-1,4-linkages. Whereas the ␤-linked residues are in the D configuration, the ␣-1,3-linked galactose units are in the rare L configuration and are further modified by a 3,6-anhydro bridge (2, 3) (Fig. 1a). On the basis of x-ray fiber diffraction, optical rotation calculations, and solution gel transition, both a parallel double helix as well as a single helix structure for agarose have been proposed, where the individual polysaccharide chains have a left-handed 3-fold helix symmetry and a pitch of 1.90 nm (3, 4). Agarose forms thermo-reversible gels structured by aggregates of agarose chains. These gels exhibit unique rheological properties and are widely used as texturing agents for various applications in the food industry or as a common laboratory medium for chromatographic separation or bacterial colony growth (5).Agarases are the glycoside hydrolases (GH) 1 that hydrolyze agarose. The ␤-agarases and the ␣-agarases cleave the internal ␤-1,4-and ␣-1,3-linkage of agarose, respectively. ␤-Agarases produce agaro-oligosaccharides in the series homologous to neoagarobiose (O-3,6-anhydro-␣-L-galact...

Polysaccharide-degrading enzymes are generally modular proteins that contain non-catalytic carbohydrate-binding modules (CBMs), which potentiate the activity of the catalytic module. CBMs have been grouped into sequence-based families, and three-dimensional structural data are available for half of these families. Clostridium thermocellum xylanase 11A is a modular enzyme that contains a CBM from family 6 (CBM6), for which no structural data are available. We have determined the crystal structure of this module to a resolution of 2.1 Å. The protein is a ␤-sandwich that contains two potential ligand-binding clefts designated cleft A and B. The CBM interacts primarily with xylan, and NMR spectroscopy coupled with site-directed mutagenesis identified cleft A, containing Trp-92, Tyr-34, and Asn-120, as the ligand-binding site. The overall fold of CBM6 is similar to proteins in CBM families 4 and 22, although surprisingly the ligand-binding site in CBM4 and CBM22 is equivalent to cleft B in CBM6. These structural data define a superfamily of CBMs, comprising CBM4, CBM6, and CBM22, and demonstrate that, although CBMs have evolved from a relatively small number of ancestors, the structural elements involved in ligand recognition have been assembled at different locations on the ancestral scaffold.

Two beta-agarase genes, agaA and agaB, were functionally cloned from the marine bacterium Zobellia galactanivorans. The agaA and agaB genes encode proteins of 539 and 353 amino acids respectively, with theoretical masses of 60 and 40 kDa. These two beta-agarases feature homologous catalytic domains belonging to family GH-16. However, AgaA displays a modular architecture, consisting of the catalytic domain (AgaAc) and two C-terminal domains of unknown function which are processed during secretion of the enzyme. In contrast, AgaB is composed of the catalytic module and a signal peptide similar to the N-terminal signature of prokaryotic lipoproteins, suggesting that this protein is anchored in the cytoplasmic membrane. Gel filtration and electrospray MS experiments demonstrate that AgaB is a dimer in solution, while AgaAc is a monomeric protein. AgaAc and AgaB were overexpressed in Escherichia coli and purified to homogeneity. Both enzymes cleave the beta-(1-->4) linkages of agarose in a random manner and with retention of the anomeric configuration. Although they behave similarly towards liquid agarose, AgaAc is more efficient than AgaB in the degradation of agarose gels. Given these organizational and catalytic differences, we propose that, reminiscent of the agarolytic system of Pseudoalteromonas atlantica, AgaA is specialized in the initial attack on solid-phase agarose, while AgaB is involved with the degradation of agarose fragments.