In agarolytic microorganisms, alpha-neoagarobiose hydrolase (NABH) is an essential enzyme to metabolize agar because it converts alpha-neoagarobiose (0-3,6-anhydro-alpha-L-galactopyranosyl-(1,3)-D-galactose) into fermentable monosaccharides (D-galactose and 3,6-anhydro-L-galactose) in the agarolytic pathway. NABH can be divided into two biological classes by its cellular location. Here, we describe a structure and function of cytosolic NABH from Saccharophagus degradans 2-40 in a native protein and D-galactose complex determined at 2.0 and 1.55 A. respectively. The overall fold is organized in an N-terminal helical extension and a C-terminal five-bladed beta-propeller catalytic domain. The structure of the enzyme-ligand (D-galactose) complex predicts a +1 subsite in the substrate binding pocket. The structural features may provide insights for the evolution and classification of NABH in agarolytic pathways. (C) 2011 Elsevier Inc. All rights reserved.