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Thioesterases are enzymes that hydrolyze thioester bonds in numerous biochemical pathways, for example in fatty acid synthesis. This work reports known functions, structures, and mechanisms of updated thioesterase enzyme families, which are classified into 35 families based on sequence similarity. Each thioesterase family is based on at least one experimentally characterized enzyme, and most families have enzymes that have been crystallized and their tertiary structure resolved. Classifying thioesterases into families allows to predict tertiary structures and infer catalytic residues and mechanisms of all sequences in a family, which is particularly useful because the majority of known protein sequence have no experimental characterization. Phylogenetic analysis of experimentally characterized thioesterases that have structures with the two main structural folds reveal convergent and divergent evolution. Based on tertiary structure superimposition, catalytic residues are predicted., (© 2021 The Protein Society.)

All acyl carrier protein primary and tertiary structures were gathered into the ThYme database. They are classified into 16 families by amino acid sequence similarity, with members of the different families having sequences with statistically highly significant differences. These classifications are supported by tertiary structure superposition analysis. Tertiary structures from a number of families are very similar, suggesting that these families may come from a single distant ancestor. Normal vibrational mode analysis was conducted on experimentally determined freestanding structures, showing greater fluctuations at chain termini and loops than in most helices. Their modes overlap more so within families than between different families. The tertiary structures of three acyl carrier protein families that lacked any known structures were predicted as well., (Copyright © 2012 The Protein Society.)

Ketoacyl synthases (KSs) catalyze condensing reactions combining acyl-CoA or acyl-acyl carrier protein (acyl-ACP) with malonyl-CoA to form 3-ketoacyl-CoA or with malonyl-ACP to form 3-ketoacyl-ACP. In each case, the resulting acyl chain is two carbon atoms longer than before, and CO2 and either CoA or ACP are formed. KSs also join other activated molecules in the polyketide synthesis cycle. Our classification of KSs by their primary and tertiary structures instead of by their substrates and the reactions that they catalyze enhances insights into this enzyme group. KSs fall into five families separated by their characteristic primary structures, each having members with the same catalytic residues, mechanisms, and tertiary structures. KS1 members, overwhelmingly named 3-ketoacyl-ACP synthase III or its variants, are produced predominantly by bacteria. Members of KS2 are mainly produced by plants, and they are usually long-chain fatty acid elongases/condensing enzymes and 3-ketoacyl-CoA synthases. KS3, a very large family, is composed of bacterial and eukaryotic 3-ketoacyl-ACP synthases I and II, often found in multidomain fatty acid and polyketide synthases. Most of the chalcone synthases, stilbene synthases, and naringenin-chalcone synthases in KS4 are from eukaryota. KS5 members are all from eukaryota, most are produced by animals, and they are mainly fatty acid elongases. All families except KS3 are split into subfamilies whose members have statistically significant differences in their primary structures. KS1 through KS4 appear to be part of the same clan. KS sequences, tertiary structures, and family classifications are available on the continuously updated ThYme (Thioester-active enzYme) database.

Thioesterases (TEs) are classified into EC 3.1.2.1 through EC 3.1.2.27 based on their activities on different substrates, with many remaining unclassified (EC 3.1.2.-). Analysis of primary and tertiary structures of known TEs casts a new light on this enzyme group. We used strong primary sequence conservation based on experimentally proved proteins as the main criterion, followed by verification with tertiary structure superpositions, mechanisms, and catalytic residue positions, to accurately define TE families. At present, TEs fall into 23 families almost completely unrelated to each other by primary structure. It is assumed that all members of the same family have essentially the same tertiary structure; however, TEs in different families can have markedly different folds and mechanisms. Conversely, the latter sometimes have very similar tertiary structures and catalytic mechanisms despite being only slightly or not at all related by primary structure, indicating that they have common distant ancestors and can be grouped into clans. At present, four clans encompass 12 TE families. The new constantly updated ThYme (Thioester-active enzYmes) database contains TE primary and tertiary structures, classified into families and clans that are different from those currently found in the literature or in other databases. We review all types of TEs, including those cleaving CoA, ACP, glutathione, and other protein molecules, and we discuss their structures, functions, and mechanisms.