The modification of thioesterase specificity has proven to be useful for genetic engineering of plants containing high levels of commercially-useful fatty acids. For example, expression of a thioesterase from the California Bay Laurel (Umbellularia californica
) in canola allowed the commercial production of a genetically engineered oil crop containing large amounts of laurate [5
] while expression of a thioesterase from Garcinia mangostana
in canola resulted in seeds containing increased amounts of stearate [22
Using an approach that compares the sequences of homologous enzymes with different substrate specificities, the substrate specificity of plant thioesterases has been shown to be mutable. However, the large number of amino acid differences between any two homologous TEs makes it difficult to identify the subset of amino acid changes that will result in a change in specificity. One commonly used approach to reduce the number of possible SDPs is to generate chimeric enzymes [9
]. Using this approach, it was found that the normally high 12:0 specificity of the Umbellularia californica
FatB enzyme can be switched to 14:0 by three amino acid changes (M197R/R199H/T231K) [9
]. However, oftentimes the resulting chimeric enzymes are either inactive or exhibit no change in specificity [8
]. What would be helpful is a method that allows the reduction of the possible SDPs to a manageable, ranked set where each change can be individually examined experimentally.
We previously reported on the Conserved Property Difference Locator (CPDL) which was designed for use in such situations [12
]. CPDL uses as input the amino acid sequence alignment of a group of enzymes broken into two homologous classes and then flags positions where there is a difference in either amino acid sequence or a property such as hydrophobicity [12
]. From the alignment of FatA versus FatB TEs, CPDL identified several potential specificity-determining positions. We chose to use the most stringent CPDL criteria and therefore individually engineered into the parent enzyme the six most dramatic changes, including five non-conservative changes and one position with a difference in amino acid charge between FatAs and FatBs.
Interestingly, four of the five residues flagged with red hourglasses identified by CPDL as putative specificity-determining positions (74, 110, 141, 174) are located in a structural element referred to as the N-terminal hot dog domain [11
]. Through the construction of chimeric enzymes, this region has been shown to control specificity [9
]. The remaining position flagged by a red hourglass (221) is near the catalytic asparagine and histidine in the second hot dog domain. However, only one of the four residues flagged with black (conservative) hourglasses identified by CPDL is in the N-terminal hot dog domain, lending validity to the selection of sites that contain conservative versus non-conservative substitutions between classes as a criterion for ranking putative specificity determining positions.
Each of these six changes suggested by CPDL were individually engineered into the parent FatB enzyme and the effect of the change was determined experimentally. Mutations at each CPDL-identified position substantially affected thioesterase activity and/or specificity. Two of the six (V110T and W221R) essentially inactivated the enzyme while the other four mutations affected substrate specificity to some degree. It is interesting to note that unlike previous studies [9
], combinations of mutations at multiple CPDL-identified positions (variants 3-MUT and 4-MUT) did not improve enzymatic performance and in fact, came close to eliminating activity.
We recently modeled the predicted structure of the plant acyl-ACP thioesterases [11
]. Using this model, we mapped the CPDL mutations relative to the predicted active site of the thioesterase (Figure ). Each of the positions is located within 16 Angstroms of the nearest catalytic residue. The monomer of the enzyme itself in the structural model is ~43 × ~49 × ~35 Angstroms. Mutations at the two positions farthest away from the catalytic site (141 and 86) have been shown to affect enzyme specificity (here and [9
]). Mutations at the two positions closest to the catalytic site (each ~8 Angstroms away) essentially inactivated the enzyme (110 and 221). Position 141 shifted specificity toward 16:1 in vivo
and is located on one of the b-sheets. Position 184, which caused a shift in specificity from 14:0 and 16:1 toward 16:0 and 18:1, is located 11 Angstroms from its closest catalytic neighbor and is on a flexible loop that has the potential to shift during binding and/or catalysis. Because this is a flexible region, there is some uncertainty regarding its conformation in the FatB structure relative to the FatB model based on threading onto the 1BVQ structure [11
3D structural model of the AtFatB enzyme . 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.
Many characteristic properties of the amino acid residues present at the CPDL-identified positions are also different between the classes. To summarize these changes, the alanine is smaller than the methionine at position 74, the threonine is smaller than methionine and has an OH group at position 141, the lysine to glutamine change at position 86 removes a positive charge and adds an amine group, the serine to glutamine change at position 174 removes an OH and adds and amine, the valine to threonine change at position 110 adds an OH, and the tryptophan to arginine change at position 221 removes a bulky aromatic side chain and adds a positive charge. The net affect of these changes appears to be a widening of the substrate binding pocket in FatA as compared to FatB (see Figure ).
The results presented here further demonstrate the viability of a sequence based approach as opposed to a more time consuming and complicated approach based on x-ray crystallography. Development of the CPDL tool facilitated a sequence-based bioinformatics approach to engineering plant acyl-ACP thioesterases for alterations in substrate specificity. Furthermore, CPDL analysis provides a straightforward method for generating hypotheses that can readily be tested regarding specificity determining positions within enzymes.