De novo fatty acid biosynthesis is terminated by the enzyme acyl-ACP thioesterase (TE), which hydrolyzes the thioester bond of the elongating acyl-ACP intermediate, and releases free fatty acids as the product of the process. The substrate specificity of acyl-ACP TE plays a critical role in determining the chain length of fatty acids produced by fatty acid synthase (FAS). The structural basis of this substrate specificity was explored via a domain-shuffling strategy using two acyl-ACP TEs (CvFatB1 and CvFatB2) from Cuphea viscosissima that share high degree of amino acid sequence homology, but express distinct substrate specificities. Two rounds of domain shuffling enabled the identification of a 41-residue region between residue 208 and 248 of CvFatB2, called Fragment III, as the most significant determinant for the substrate specificity. Based on a multiple sequence alignment of 21 functionally characterized plant acyl-ACP TEs, nine residues were predicted to affect the substrate specificity of these enzymes. Together 15 specific residues of CvFatB2 were subjected to site-directed mutagenesis analysis to verify their effects on substrate specificity. These analyses demonstrate that six specific residues play critical roles in determining the substrate specificity of CvFatB2, including V194, V217, N223, R226, R227, and I268. Another three residues, L257, I260, and L289, proved to impact the catalytic activity of acyl-ACP TE, because they are in two proposed ACP binding motifs. Analysis of a crystal structure of acyl-ACP TE from Lactobacillus plantarum (2OWN) and comparing that with the experimental data in this study led to a hypothesis that acyl-ACP TEs have a cavity in the N-terminal hotdog domain between the central a-helix and the anti-parallel �-sheets, and the depth of this cavity determines the substrate specificity of the enzyme.

Dissecting the structural determinants of chain length selectivity of acyl-ACP thioesterases

248th National Meeting (2014)

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