Identification of amino acid residues involved in substrate specificity of plant acyl-ACP thioesterases using a bioinformatics-guided approach

Abstract

Background: The large amount of available sequence information for the plant acyl-ACP thioesterases (TEs) made it possible to use a bioinformatics-guided approach to identify amino acid residues involved in substrate specificity. The Conserved Property Difference Locator (CPDL) program allowed the identification of putative specificity-determining residues that differ between the FatA and FatB TE classes. Six of the FatA residue differences identified by CPDL were incorporated into the FatB-like parent via site-directed mutagenesis and the effect of each on TE activity was determined. Variants were expressed in E. coli strain K27 that allows determination of enzyme activity by GCMS analysis of fatty acids released into the medium.

Results: Substitutions at four of the positions (74, 86, 141, and 174) changed substrate specificity to varying degrees while changes at the remaining two positions, 110 and 221, essentially inactivated the thioesterase. The effects of substitutions at positions 74, 141, and 174 (3-MUT) or 74, 86, 141, 174 (4-MUT) were not additive with respect to specificity.

Conclusion: Four of six putative specificity determining positions in plant TEs, identified with the use of CPDL, were validated experimentally; a novel colorimetric screen that discriminates between active and inactive TEs is also presented.

Figure 1

The FatA and FatB classes of plant acyl-ACP thioesterases. Enzymes in the FatA class are active on 18:1-ACP while those in the FatB class are active on saturated fatty acids of various chain lengths. Only enzymes whose substrate specificity has been demonstrated experimentally are shown. The NCBI accession numbers are provided in the figure. Numbers in the enzyme name were designated by depositors, except in the case of AtFatA3 and AtFatA4 where the number refers to the chromosome location of the gene as a way to distinguish between the sequences. At, Arabidopsis thaliana; Bj, Bradyrhizobium japonicum; Bn, Brassica napus; Cc, Cinnamonum camphorum; Cch, Capsicum chinense; Ch, Cuphea hookeriana; Cl, Cuphea lanceolata; Cp, Cuphea palustris; Cs, Coriandrum sativum; Ct, Carthamus tinctorius; Cw,Cuphea wrightii; Eg, Elaeis guineensis; Gh, Gossypium hirsutum; Gm, Garcinia mangostana; Ha, Helianthus annuus; Ig, Iris germanica; It, Iris tectorum; Mf, Myristica fragrans; Ta, Triticum aestivum; Ua, Ulmus americana; Uc, Umbellularia californica.

Figure 2

Amino acid sequence alignment of Arabidopsis thaliana FatA3 (NP_189147) with FatB3-2 (CAA85388). The first residue of the mature enzyme is marked with an arrow. The two hot-dog domains of the 3D structural model [11] are underlined. Completely conserved residues are underlined. Amino acid positions determined to be SDPs by previous authors are marked with filled circles. Asterisks denote the CPDL-identified putative specificity determining positions. + marks residues comprising the catalytic triad of C, H, N. X's mark positions where mutations inactivated the enzyme.

Figure 3

A portion of the CPDL [12] output for the FatA (upper) versus FatB (lower) alignment. Arrows denote residues that are conserved in all or "all-but-one" of the sequences in each class. One of the putative SDPs examined in this study (W221R) is flagged with a red hourglass.

Figure 4

(A) The total fatty acid content (nmol/ml) in the medium from E. coli clones containing the variants listed as determined by GCMS of FAMEs. (B) Quantity of each fatty acid present in each of the variants. Error bars represent the standard error for five independent clones of each variant.

Figure 5

MacConkey agar plate-based screen for plant thioesterase activity. Colonies that contain an active thioesterase variant are white while those containing either empty vector (pBC) or an inactive variant are dark pink. Colonies exhibiting a range of activities could be reliably screened visually with this assay.

Figure 6

3D structural model of the AtFatB enzyme [11]. The CPDL-identified residues are shown in blue. The catalytic triad is circled with the residues colored red. The substrate from the bacterial enzyme is shown in orange for reference.