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

Jing, Fuyuan; Zhao, Le; Yandeau‐Nelson, Marna D.; Nikolau, Basil J.

doi:10.1038/s41467-018-03310-z

Cited by 37 publications

(57 citation statements)

References 27 publications

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“…Residues involved in the substrate binding pocket and catalysis ( Feng et al, 2017 ) are highlighted in black and gray, respectively. Positively charged surface patches ( Serrano-Vega et al, 2005 ; Jing et al, 2018 ), and the N- and C-terminal domains are boxed in blue and black, respectively.…”

Section: Resultsmentioning

confidence: 99%

“…Structural models have been proposed for Ha FatA ( Serrano-Vega et al, 2005 ), and for FatB from Arabidopsis thaliana and Cuphea viscosissima ( Mayer and Shanklin, 2005 ; Jing et al, 2018 ), and more recently, the crystal structure of FatB1 from Umbellularia californica was determined by X-ray diffraction (PDB 5X04.A: Feng et al, 2017 ). The amino acid sequence, excluding the signal peptide region, of this Uc FatB was compared to the Ha FatA (residues 89–361) and Ha FatB (residues 129–414) sequences, showing 44.57 and 58.05% identity, respectively ( Figure 2 ).…”

Section: Resultsmentioning

confidence: 99%

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New Insights Into Sunflower (Helianthus annuus L.) FatA and FatB Thioesterases, Their Regulation, Structure and Distribution

et al. 2018

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Sunflower seeds (Helianthus annuus L.) accumulate large quantities of triacylglycerols (TAG) between 12 and 28 days after flowering (DAF). This is the period of maximal acyl-acyl carrier protein (acyl-ACP) thioesterase activity in vitro, the enzymes that terminate the process of de novo fatty acid synthesis by catalyzing the hydrolysis of the acyl-ACPs synthesized by fatty acid synthase. Fatty acid thioesterases can be classified into two families with distinct substrate specificities, namely FatA and FatB. Here, some new aspects of these enzymes have been studied, assessing how both enzymes contribute to the acyl composition of sunflower oil, not least through the changes in their expression during the process of seed filling. Moreover, the binding pockets of these enzymes were modeled based on new data from plant thioesterases, revealing important differences in their volume and geometry. Finally, the subcellular location of the two enzymes was evaluated and while both possess an N-terminal plastid transit peptide, only in FatB contains a hydrophobic sequence that could potentially serve as a transmembrane domain. Indeed, using in vivo imaging and organelle fractionation, H. annuus thioesterases, HaFatA and HaFatB, appear to be differentially localized in the plastid stroma and membrane envelope, respectively. The divergent roles fulfilled by HaFatA and HaFatB in oil biosynthesis are discussed in the light of our data.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

New Insights Into Sunflower (Helianthus annuus L.) FatA and FatB Thioesterases, Their Regulation, Structure and Distribution

et al. 2018

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“…The six highly conserved residues (Trp192, Asp309, Asn311, His313, Tyr319, and Glu347) identified in the multiple sequence alignment are similarly oriented in the L. plantarum TE structure and in the homology model of CvFatB2 (Figure 2). One of these residues, Trp192 in CvFatB2, that resides in the N-terminal hotdog domain and is far removed from the active site, has been recently shown to determine the cavity-size that binds the acyl-moiety of the acyl-ACP substrate [24]. The other conserved residues (Asp309, Asn311, His313, Tyr319, and Glu347 in CvFatB2) are in the C-terminal hotdog domain.…”

Section: Predicted Structure Of a Plant Acyl-acp Tementioning

confidence: 99%

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

Jing

Yandeau‐Nelson

Nikolau

2018

Biochemical Journal

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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.

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“…Two distinct regions in the N-terminal domain of these enzymes have recently been shown to influence substrate specificity. A hydrophobic cavity binds the acyl moiety of acyl-ACP substrates and dictates preference for particular acyl chain lengths, and a conserved RKLXKX motif creates a positively charged surface region thought to facilitate ACP binding and fatty acyl chain extrusion into the thioesterase's hydrophobic cavity for subsequent cleavage (Jing et al, 2018;Ziesack et al, 2018).…”

Section: Roles Of Acyl-acp Thioesterases In Specialized Metabolismmentioning

confidence: 99%

“…Low activity in heterologous hosts and lack of selectivity for substrates of the desired fatty acyl chain length are key limitations to the use of heterologously expressed plant acyl-ACP thioesterases as sustainable sources of fatty acids and their chemical derivatives (Hernández Lozada et al, 2018). Genetic selection strategies, domain-swapping approaches, and structure-guided mutagenesis based on computational redesign, have all been used to increase the activity of plant thioesterases or direct their substrate preferences toward acyl-ACP chain lengths that suit specific industrial purposes (Feng et al, 2017;Grisewood et al, 2017;Hernández Lozada et al, 2018;Jing et al, 2018;Ziesack et al, 2018). Given the natural substrate specificities of ALT enzymes, recombinant ALT enzymes show promise as components of biological systems for renewable methyl ketone production.…”

Section: Engineering Recombinant Fatty Acyl-acp Thioesterases and Fatmentioning

confidence: 99%

Fatty Acyl Synthetases and Thioesterases in Plant Lipid Metabolism: Diverse Functions and Biotechnological Applications

Kalinger

Pulsifer

Hepworth

et al. 2020

Lipids

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Plants use fatty acids to synthesize acyl lipids for many different cellular, physiological, and defensive roles. These roles include the synthesis of essential membrane, storage, or surface lipids, as well as the production of various fatty acid-derived metabolites used for signaling or defense. Fatty acids are activated for metabolic processing via a thioester linkage to either coenzyme A or acyl carrier protein. Acyl synthetases metabolically activate fatty acids to their thioester forms, and acyl thioesterases deactivate fatty acyl thioesters to free fatty acids by hydrolysis. These two enzyme classes therefore play critical roles in lipid metabolism. This review highlights the surprisingly complex and varying roles of fatty acyl synthetases in plant lipid metabolism, including roles in the intracellular trafficking of fatty acids. This review also surveys the many specialized fatty acyl thioesterases characterized to date in plants, which produce a great diversity of fatty acid products in a tissue-specific manner. While some acyl thioesterases produce fatty acids that clearly play roles in plant-insect or plant-microbial interactions, most plant acyl thioesterases have yet to be fully characterized both in terms of their substrate specificities and their functions. The biotechnological applications of plant acyl thioesterases and synthetases are also discussed, as there is significant interest in these enzymes as catalysts for the sustainable production of fatty acids and their derivatives for industrial uses.

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

Cited by 37 publications

References 27 publications

New Insights Into Sunflower (Helianthus annuus L.) FatA and FatB Thioesterases, Their Regulation, Structure and Distribution

New Insights Into Sunflower (Helianthus annuus L.) FatA and FatB Thioesterases, Their Regulation, Structure and Distribution

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

Fatty Acyl Synthetases and Thioesterases in Plant Lipid Metabolism: Diverse Functions and Biotechnological Applications

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