Structural Insight into Acyl-ACP Thioesterase toward Substrate Specificity Design

Abstract

Acyl-ACP thioesterase (TE) catalyzes the hydrolysis of thioester bonds during type II fatty acid synthesis and directly determines fatty acid chain length. Most TEs are responsible for recognition of 16:0 and 18:1 substrates, while specific TEs interrupt acyl-ACP elongation at C8-C14. However, the acyl selection mechanism of TE has not been thoroughly elucidated to date. In this study, the crystal structure of the C12-specific thioesterase FatB from Umbellularia californica, which consists of two independent hotdog domains, was determined. An uncanonical Asp-His-Glu catalytic network was identified on the C-terminal hotdog domain, whereas the substrate binding pocket was determined to be on the N-terminal hotdog domain. Moreover, we elucidated UcFatB's substrate selection mechanism, which is accommodated by several unconservative amino acids on the β5, β2, and β4 sheets and enclosed by T137 on the α1 helix. On this basis, the C12-specific TE was rationally redesigned toward C14 selectivity by tuning the substrate binding pocket capacity. The T137G mutant demonstrated comparative relative activity on C14 substrates compared to C12 substrates in vitro. Furthermore, the reconstructed UcFatB_T137G achieved C14 fatty acid content up to 40% in contrast to 10% C14 from the wild type in engineered E. coli cells. The unraveled substrate selection mechanism of TE provides a new strategy for tailoring fatty acid synthesis.

Fatty Acid Biosynthesis: Chain‐Length Regulation and Control

“…Thr137 is located near the bottom of the binding pocket and its bulky side chain restricts binding of acyl chains longer than C 12 , thus making lauric acid (C 12 ) the main product. In contrast, the rationally designed mutant Thr137Gly showed a product spectrum shifted towards longer fatty acids of C 14 –C 16 in length …”

“…The best‐characterized plant TE is FatB from Umbellularia californica (termed UcFatB), which has been extensively studied for almost three decades . Its X‐ray structure having recently been determined (PDB ID: 5X04) . UcFatB forms a homodimer, each monomer showing a tandem hotdog fold.…”

“…[9,116] Its X-ray structure havingr ecently been determined (PDB ID:5 X04). [117] UcFatB forms ah omodimer,e ach monomer showing at andem hotdogf old. Structurala nalysisa nd site-directed mutagenesis revealed the substrate-binding pocket and residues Asp281, Asn283, His285, and Glu319 forming the catalytic network.…”

“…In contrast, the rationally designed mutant Thr137Gly showedaproduct spectrums hiftedt owards longerf atty acids of C 14 -C 16 in length. [117] ChemBioChem 2019, 20,2298 -2321 www.chembiochem.org…”

Overexpression of Cuphea viscosissima CvFatB4 enhances 16:0 fatty acid accumulation in Arabidopsis

“…1) (Emanuelsson et al 2007). Sequence alignment of the deduced amino acid sequence of CvFatB4 and several plant FatB proteins with known biochemical activities revealed that four amino acid residues (Asp 312 , Asn 314 , His315, and Glu 350 in CvFatB4) are conserved and localized in so-called hot-dog folds, suggesting that they are catalytically important and CvFatB4 catalyzes the hydrolysis of the thioester bond using the catalytic mechanism found in UcFatB (Dani et al 2011;Dörmann et al 1995;Feng et al 2017; Sánchez-García et al 2010).…”