3,6-Anydro-L-galatonate cycloisomerase (ACI) catalyzes the cycloisomerization of a 3,6-anhydro-c-galactonic acid known as a novel metabolite in agarolytic bacteria. Here, we present 3-D structures of ACI from Vibrio sp. strain EJY3 (VejACI) in native and mutant forms at 2.2 angstrom and 2.6 angstrom resolutions, respectively. The enzyme belongs to the mandelate racemase subgroup of the enolase superfamily catalyzing common beta-elimination reactions by alpha-carbon deprotonation of substrates. The structure of VejACI revealed a notable 20s loop region in the capping domain, which can be a highly conserved structural motif in ACI homologs of agar metabolism. By comparing mutant (mVejAC/H300 N) and native VejACI structures, we identified a conformational change of I1e142 in VejACI that causes spatial expansion in the binding pocket. These observations imply that I1e142 and the 20s loop play important roles in enzymatic reactivity and substrate specificity. The structural phylogenetic analysis of the enolase superfamily including ACIs revealed sequential, structural, and functional relationships related to the emergence of novel substrate specificity. (C) 2017 Elsevier Inc. All rights reserved.