Two distinct domains contribute to the substrate acyl chain length selectivity of plant acyl-ACP thioesterase

Fuyuan Jing,Basil J Nikolau,Le Zhao,Marna D Yandeau-Nelson

doi:10.1038/s41467-018-03310-z

Abstract

The substrate specificity of acyl-ACP thioesterase (TE) plays an essential role in controlling the fatty acid profile produced by type II fatty acid synthases. Here we identify two groups of residues that synergistically determine different substrate specificities of two acyl-ACP TEs from Cuphea viscosissima (CvFatB1 and CvFatB2). One group (V194, V217, N223, R226, R227, and I268 in CvFatB2) is critical in determining the structure and depth of a hydrophobic cavity in the N-terminal hotdog domain that binds the substrate’s acyl moiety. The other group (255-RKLSKI-260 and 285-RKLPKL-289 in CvFatB2) defines positively charged surface patches that may facilitate binding of the ACP moiety. Mutagenesis of residues within these two groups results in distinct synthetic acyl-ACP TEs that efficiently hydrolyze substrates with even shorter chains (C4- to C8-ACPs). These insights into structural determinants of acyl-ACP TE substrate specificity are useful in modifying this enzyme for tailored fatty acid production in engineered organisms.

Highlights

The substrate specificity of acyl-ACP thioesterase (TE) plays an essential role in controlling the fatty acid profile produced by type II fatty acid synthases
The biosynthesis of Fatty acids (FAs) is catalyzed by FA synthases (FAS) that occur in two ternary forms, the multicomponent type II FAS that occurs in bacteria and plants[3], and the multifunctional type I FAS that occurs in fungi and animals[4]
Sequence identity, but when expressed in E. coli, CvFatB2 acts on 14- and 16-carbon fatty acyl-ACPs, whereas CvFatB1 acts on 8and 10-carbon fatty acyl-ACPs10

Summary

Introduction

The substrate specificity of acyl-ACP thioesterase (TE) plays an essential role in controlling the fatty acid profile produced by type II fatty acid synthases. Mutagenesis of residues within these two groups results in distinct synthetic acyl-ACP TEs that efficiently hydrolyze substrates with even shorter chains (C4- to C8-ACPs) These insights into structural determinants of acyl-ACP TE substrate specificity are useful in modifying this enzyme for tailored fatty acid production in engineered organisms. The industrial application of FAs is determined by two attributes, their carbon chain length and the degree of unsaturation The former attribute is enzymologically determined by the substrate specificity of the acyl-ACP thioesterase (TE) that catalyzes the terminal reaction of the type II FAS system. The biotechnological focus on acyl-ACP TEs was primed by the discovery that this enzyme is the major determinant that enables seeds of certain plants (e.g., California Bay Laurel tree, and members of the Palmae family) to produce laurate-rich oils[5,6] This trait was subsequently transgenically transferred to annual crops, such as oilseed rape, resulting in the accumulation of seed oil containing over 50% of lauric acid[7]. These characterizations led to the creation of more than 60 synthetic enzymes, some of which have acquired catalytic capabilities that may have relevance to the more efficient conversion of sugar-derived carbon to energy-dense molecules that have applications as biofuels or bioproducts

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Conclusion