Characterization of substrate specificity of plant FatA and FatB acyl-ACP thioesterases

https://doi.org/10.1016/S0003-9861(02)00017-6Get rights and content

Abstract

The specificity of plant acyl–acyl carrier protein (ACP) thioesterases is the major determinant of the chain length and level of saturated fatty acids found in most plant tissues. Although these enzymes have been previously characterized from a number of sources, information on kinetic parameters for a wide range of substrates with cloned enzymes is lacking. In the present study the substrate specificity of recombinant FatA thioesterase isoforms from Arabidopsis (AtFatA) and coriander (CsFatA) and FatB from Arabidopsis (AtFatB) have been re-examined with a comprehensive range of substrates including 14:1-ACP and 16:1-ACP. AtFatA displayed the highest catalytic efficiencies (kcat/Km) towards oleoyl-ACP with activities at least 20-fold lower for all other tested substrates and 75-fold lower with palmitoyl-ACP. Both chain length and double bond presence strongly influenced kcat of FatA with minor influence on Km. Arabidopsis FatB substrate specificity was found to differ from previous reports and this difference could be attributed to the influence of ACP structure. FatB activity with palmitoyl-ACP was 2.5-fold higher and the ratio of 16:0-ACP/14:0-ACP hydrolysis was 6.4-fold higher with spinach ACP compared to E. coli ACP. Additionally, the influence of amino acid domains from both AtFatA and AtFatB on their substrate specificity was studied by utilizing a domain-swapping approach. The characterization of the resulting chimeric enzymes pointed to the N-terminus as a determinant of the substrate specificity for both FatA and FatB acyl-ACP thioesterases.

Section snippets

Experimental

Preparation of acyl-ACP substrates. Labeled acyl-ACP substrates were prepared using a recombinant acyl-ACP synthetase from E. coli kindly provided by John Shanklin (Brookhaven National Laboratory). Acylation reactions contained 50 μg of recombinant ACP-I from spinach, 660 Mbeq (approximately 0.1 μmol) of [1-14C] fatty acid ammonium salt ([3H] fatty acid in the case of 16:1Δ9), 5 mM ATP, 2 mM DTT, 4 mM LiCl2, 10 mM MgCl2, 100 mM Tris pH 8.0, and 10 μg acyl-ACP synthetase in a final volume of 0.5

Results and discussion

Although the substrate specificity of plant acyl-ACP thioesterases has previously been examined from several species [30] most of such studies had one or more limitations. First, analysis of plant extracts can be complicated by the presence of both FatA and FatB activities or by multiple isoforms of each enzyme. Second, because of limited quantities of available radiolabeled acyl-ACP substrates, very few studies have included kinetic analysis. Third, the number of substrates tested has been

Acknowledgements

The work was supported in part by grants from The Dow Chemical Company, Dow Agrosciences and the National Science Foundation. We also acknowledge the Michigan Agricultural Experiment Station for support. We thank John Shanklin for a gift of acyl-ACP synthase, David Schultz for supplying 14:0-ACP desaturase, and Jay thelen for critical reading of the manuscript.

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