Bioinformatics analysis was focused on unique acetylesterases annotated in the CAZy database within the CE16 family and simultaneously belonging to the SGNH hydrolase superfamily. The CE16 acetylesterases were compared to structurally related SGNH hydrolases: (i) selected members of the CE2, CE3, CE6, CE12 and CE17 family of the CAZy database and (ii) structural representatives of the Lipase_GDSL and Lipase_GDSL_2 families according to the Pfam database. Sequence alignment based on four conserved sequence regions (CSRs) containing active-site residues was used to calculate sequence logos specific for each CE family and to construct a phylogenetic tree. In many members of the CE16 family, aspartic acid from the Ser–His–Asp catalytic triad has been replaced by asparagine, and based on structure–sequence comparison, an alternative catalytic dyad mechanism was predicted for these enzymes. In addition to four conserved regions, CSR-I, CSR-II, CSR-III and CSR-V, containing catalytic and oxyanion-hole residues, CSR-IV was found in the CE16 family as the only CAZy family. Tertiary structures of the characterized CE16 members prepared by homology modeling showed that the α/β/α sandwich fold as well as the topology of their active sites are preserved. The phylogenetic tree and sequence alignment indicate the existence of a subfamily in the CE16 family fully consistent with the known biochemical data. In addition, nonstandard CE16 members that differ from others were analyzed and their active-site residues were predicted. A better understanding of the structure–function relationship of acetylesterases can help in the targeted design of these enzymes for biotechnology.

生物信息学分析的重点是在 CE16 家族内的 CAZy 数据库中注释的独特乙酰酯酶，同时属于 SGNH 水解酶超家族。将 CE16 乙酰酯酶与结构相关的 SGNH 水解酶进行了比较：（i）CAZy 数据库的 CE2、CE3、CE6、CE12 和 CE17 家族的选定成员和（ii）根据 Pfam 数据库的 Lipase_GDSL 和 Lipase_GDSL_2 家族的结构代表。基于包含活性位点残基的四个保守序列区域 (CSR) 的序列比对用于计算每个 CE 家族特异性的序列徽标并构建系统发育树。在 CE16 家族的许多成员中，来自 Ser-His-Asp 催化三联体的天冬氨酸已被天冬酰胺取代，并且基于结构-序列比较，预测了这些酶的替代催化二元机制。除了四个保守区域，CSR-I、CSR-II、CSR-III 和 CSR-V，包含催化和氧阴离子空穴残基，CSR-IV 在 CE16 家族中被发现是唯一的 CAZy 家族。通过同源建模制备的特征性 CE16 成员的三级结构表明，α/β/α 夹心折叠及其活性位点的拓扑结构得以保留。系统发育树和序列比对表明CE16家族中存在一个与已知生化数据完全一致的亚家族。此外，分析了与其他成员不同的非标准 CE16 成员，并预测了它们的活性位点残基。