<div><p>A deep-sea bacterium, <i>Microbulbifer thermotolerans</i> JAMB-A94, has a β-agarase (<i>Mt</i>AgaA) belonging to the glycoside hydrolase family (GH) 16. The optimal temperature of this bacterium for growth is 43–49 °C, and <i>Mt</i>AgaA is stable at 60 °C, which is one of the most thermostable enzymes among GH16 β-agarases. Here, we determined the catalytic domain structure of <i>Mt</i>AgaA. <i>Mt</i>AgaA consists of a β-jelly roll fold, as observed in other GH16 enzymes. The structure of <i>Mt</i>AgaA was most similar to two β-agarases from <i>Zobellia galactanivorans, Zg</i>AgaA, and <i>Zg</i>AgaB. Although the catalytic cleft structure of <i>Mt</i>AgaA was similar to <i>Zg</i>AgaA and <i>Zg</i>AgaB, residues at subsite −4 of <i>Mt</i>AgaA were not conserved between them. Also, an α-helix, designated as α4′, was uniquely located near the catalytic cleft of <i>Mt</i>AgaA. A comparison of the structures of the three enzymes suggested that multiple factors, including increased numbers of arginine and proline residues, could contribute to the thermostability of <i>Mt</i>AgaA.</p></div
