Identification of active site residues implies a two-step catalytic mechanism for acyl-ACP thioesterase

Abstract

In plants and bacteria that use a Type II fatty acid synthase, isozymes of acyl-acyl carrier protein (ACP) thioesterase (TE) hydrolyze the thioester bond of acyl-ACPs, terminating the process of fatty acid biosynthesis. These TEs are therefore critical in determining the fatty acid profiles produced by these organisms. Past characterizations of a limited number of plant-sourced acyl-ACP TEs have suggested a thiol-based, papain-like catalytic mechanism, involving a triad of Cys, His, and Asn residues. In the present study, the sequence alignment of 1019 plant and bacterial acyl-ACP TEs revealed that the previously proposed Cys catalytic residue is not universally conserved and therefore may not be a catalytic residue. Systematic mutagenesis of this residue to either Ser or Ala in three plant acyl-ACP TEs, CvFatB1 and CvFatB2 from Cuphea viscosissima and CnFatB2 from Cocos nucifera, resulted in enzymatically active variants, demonstrating that this Cys residue (Cys348 in CvFatB2) is not catalytic. In contrast, the multiple sequence alignment, together with the structure modeling of CvFatB2, suggests that the highly conserved Asp309 and Glu347, in addition to previously proposed Asn311 and His313, may be involved in catalysis. The substantial loss of catalytic competence associated with site-directed mutants at these positions confirmed the involvement of these residues in catalysis. By comparing the structures of acyl-ACP TE and the Pseudomonas 4-hydroxybenzoyl-CoA TE, both of which fold in the same hotdog tertiary structure and catalyze the hydrolysis reaction of thioester bond, we have proposed a two-step catalytic mechanism for acyl-ACP TE that involves an enzyme-bound anhydride intermediate.

Identification and Functional Characterization of a Soybean (Glycine max) Thioesterase that Acts on Intermediates of Fatty Acid Biosynthesis

“…However, the well-known and widely used approach of analyzing fatty acid components produced by thioesterases heterologously expressed in E. coli provided an efficient and fast way to study the in vivo activity of these enzymes. In this experimental system, we were not able to observe 3-ketoacids directly but rather assumed their presence on the basis of the detection of 2-methylketones following chemical decarboxylation of the 3-ketoacids [9,30]. GmMKS2-X2, for which the corresponding cDNA was isolated from the immature seeds of G. max, produced primarily 2-tridecenone (13:1) and smaller amounts of other odd-chain methylketones (7:0, 9:0, 11:0, 11:1, 13:0, 15:0, 15:1, and 17:1) when expressed in E. coli and upon heat-activated decarboxylation.…”

Structure, function, and regulation of thioesterases

“…Mutation of the predicted Cys active site residue to Ala resulted in approximately 70% reduced activity compared to wild-type, thus it is possible that this Cys residue is important for substrate binding J o u r n a l P r e -p r o o f Journal Pre-proof but not catalytic activity, or that mutation of this residue perturbs the function of the conserved adjacent Glu 319 . Furthermore, multiple sequence alignment of 1019 acyl-ACP TE sequences collected from the ThYme database revealed that Cys 320 is only conserved among 22% of the 1019 sequences, and appears restricted to plant acyl-ACP TEs [144].…”

“…Binding of the substrate positions the thioester bond adjacent to the catalytic network. Two similar mechanisms have been proposed based on the structures of TE14 thioesterases, and while they differ in the exact role of the catalytic residues, in general, both mechanisms involve a nucleophile that attacks the thioester carbonyl carbon to form an acyl-anhydride intermediate, which is subsequently hydrolysed by water molecules to release non-esterified fatty acids [141,144].…”

“…The structures of DEBS TE and PICS TE crystallised at differing pH values demonstrate pH induced conformational changes, with the lid region closing to form a long substrate channel in DEBS TE structures crystallised at a pH ≤7.5 (PDB 1KEZ, 5D3K, 5D3Z), whereas the crystal Additional pH induced conformational changes are observed in the loop region between strands β8 and β9 (residues 230-250), and the short loop between the C-terminal of strand β9 and Nterminal of helix α9 (residues 255-260). Tsai et al [144] suggest that at pH 8.5, the DEBS TE structure represents an inactive conformation. At this pH, the conformational change in the flexible lid region alters the loop between strands β8 and β9, which appears to partially unravel helix α9 and displace residues 255-260.…”

Machine learning model of the catalytic efficiency and specificity of acyl-ACP thioesterase variants generated by natural and in vitro directed evolution

“…Limited structural information and a not well-understood catalytic mechanism for acyl-ACP TEs 26,27 makes it challenging to increase the activity of this enzyme by rational design. Directed evolution, which mimics the natural evolutionary process at the lab scale, is a promising alternative approach, and provides a mechanism to identify and exploit genetic space that natural evolution may not have explored [28][29][30] .…”