Family Report for: Thioesterase_acyl-transferase

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.

The crystal structure of a myristoyl acyl carrier protein specific thioesterase (C14ACP-TE) from a bioluminescent bacterium, Vibrio harveyi, was solved by multiple isomorphous replacement methods and refined to an R factor of 22% at 2.1-A resolution. This is the first elucidation of a three-dimensional structure of a thioesterase. The overall tertiary architecture of the enzyme resembles closely the consensus fold of the rapidly expanding superfamily of alpha/beta hydrolases, although there is no detectable homology with any of its members at the amino acid sequence level. Particularly striking similarity exists between the C14ACP-TE structure and that of haloalkane dehalogenase from Xanthobacter autotrophicus. Contrary to the conclusions of earlier studies [Ferri, S. R., & Meighen, E. A. (1991) J. Biol. Chem. 266, 12852-12857] which implicated Ser77 in catalysis, the crystal structure of C14ACP-TE reveals a lipase-like catalytic triad made up of Ser114, His241, and Asp211. Surprisingly, the gamma-turn with Ser114 in a strained secondary conformation (phi = 53 degrees, psi = -127 degrees), characteristic of the so-called nucleophilic elbow, does not conform to the frequently invoked lipase/esterase consensus sequence (Gly-X-Ser-X-Gly), as the positions of both glycines are occupied by larger amino acids. Site-directed mutagenesis and radioactive labeling support the catalytic function of Ser114. Crystallographic analysis of the Ser77-->Gly mutant at 2.5-A resolution revealed no structural changes; in both cases the loop containing the residue in position 77 is disordered.

The nucleotide sequence of luxD (EMBL accession No. X65611), encoding acyltransferase (ACT), of the lux operon from Photobacterium leiognathi PL741 was determined, and the amino acid (aa) sequence was deduced. ACT is a component of the fatty acid reductase complex, which is responsible for converting fatty acid to aldehyde that serves as the substrate in the luciferase-catalyzed bioluminescent reactions. The protein has a calculated M(r) of 34,384 and comprises 305 aa residues. Alignment and comparison of the ACT of P. leiognathi with that of Vibrio fischeri ATCC7744, V. harveyi B392 and Xenorhabdus luminescens Hm shows that there is 66%, 59% and 61% aa identity, respectively.

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.

The ThYme (Thioester-active enzYme; http://www.enzyme.cbirc.iastate.edu) database has been constructed to bring together amino acid sequences and 3D (tertiary) structures of all the enzymes constituting the fatty acid synthesis and polyketide synthesis cycles. These enzymes are active on thioester-containing substrates, specifically those that are parts of the acyl-CoA synthase, acyl-CoA carboxylase, acyl transferase, ketoacyl synthase, ketoacyl reductase, hydroxyacyl dehydratase, enoyl reductase and thioesterase enzyme groups. These groups have been classified into families, members of which are similar in sequences, tertiary structures and catalytic mechanisms, implying common protein ancestry. ThYme is continually updated as sequences and tertiary structures become available.

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.

The crystal structure of a myristoyl acyl carrier protein specific thioesterase (C14ACP-TE) from a bioluminescent bacterium, Vibrio harveyi, was solved by multiple isomorphous replacement methods and refined to an R factor of 22% at 2.1-A resolution. This is the first elucidation of a three-dimensional structure of a thioesterase. The overall tertiary architecture of the enzyme resembles closely the consensus fold of the rapidly expanding superfamily of alpha/beta hydrolases, although there is no detectable homology with any of its members at the amino acid sequence level. Particularly striking similarity exists between the C14ACP-TE structure and that of haloalkane dehalogenase from Xanthobacter autotrophicus. Contrary to the conclusions of earlier studies [Ferri, S. R., & Meighen, E. A. (1991) J. Biol. Chem. 266, 12852-12857] which implicated Ser77 in catalysis, the crystal structure of C14ACP-TE reveals a lipase-like catalytic triad made up of Ser114, His241, and Asp211. Surprisingly, the gamma-turn with Ser114 in a strained secondary conformation (phi = 53 degrees, psi = -127 degrees), characteristic of the so-called nucleophilic elbow, does not conform to the frequently invoked lipase/esterase consensus sequence (Gly-X-Ser-X-Gly), as the positions of both glycines are occupied by larger amino acids. Site-directed mutagenesis and radioactive labeling support the catalytic function of Ser114. Crystallographic analysis of the Ser77-->Gly mutant at 2.5-A resolution revealed no structural changes; in both cases the loop containing the residue in position 77 is disordered.

The nucleotide sequence of luxD (EMBL accession No. X65611), encoding acyltransferase (ACT), of the lux operon from Photobacterium leiognathi PL741 was determined, and the amino acid (aa) sequence was deduced. ACT is a component of the fatty acid reductase complex, which is responsible for converting fatty acid to aldehyde that serves as the substrate in the luciferase-catalyzed bioluminescent reactions. The protein has a calculated M(r) of 34,384 and comprises 305 aa residues. Alignment and comparison of the ACT of P. leiognathi with that of Vibrio fischeri ATCC7744, V. harveyi B392 and Xenorhabdus luminescens Hm shows that there is 66%, 59% and 61% aa identity, respectively.