Alr1529, a serine hydrolase from the cyanobacteria Anabaena sp. strain PCC 7120 is a member of the SGNH hydrolase superfamily. Biochemical characterization of the purified enzyme revealed that the protein is a dimer in solution and is specific for aryl esters of short chain carboxylic acids. The enzyme was regio-selective for α-naphthyl esters with maximum activity at pH 7.5 and has a broad optimal temperature range (25-45 °C). A structure based comparison of Alr1529 with other superfamily members confirmed the presence of the catalytic triad (Ser17-Asp179-His182) and oxyanion hole (Ser17-Arg54-Asn87) residues. Alr1529 exhibits a previously undescribed variation in the active site wherein a conserved Gly, a proton donor making up the oxyanion hole in the SGNH hydrolases, is substituted by Arg54. Site-directed mutagenesis studies suggest that Arg54 is crucial for substrate binding and catalytic activity. Ser17 plays a very crucial role in catalysis as evident from the 50-fold lower activity of the S17A mutant. © 2008 Elsevier B.V. All rights reserved.