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SUMMARY Plants synthesize an array of volatile compounds, many of which serve ecological roles in attracting pollinators, deterring herbivores, and communicating with their surroundings. Methyl anthranilate (MeAA) is an anti‐herbivory defensive volatile responsible for grape aroma that is emitted by several agriculturally relevant plants, including citrus, grapes, and maize. Unlike maize, which uses a one‐step anthranilate methyltransferase (AAMT), grapes have been thought to use a two‐step pathway for MeAA biosynthesis. By mining available transcriptomics data, we identified two AAMTs inVitis vinifera(wine grape), as well as one ortholog in “Concord” grape. Many angiosperms methylate the plant hormone salicylic acid (SA) to produce methyl salicylate, which acts as a plant‐to‐plant communication molecule. Because theCitrus sinensis(sweet orange) SA methyltransferase can methylate both anthranilate (AA) and SA, we used this enzyme to examine the molecular basis of AA activity by introducing rational mutations, which identified several active site residues that increase activity with AA. Reversing this approach, we introduced mutations that imparted activity with SA in the maize AAMT, which uncovered different active site residues from those in the citrus enzyme. Sequence and phylogenetic analysis revealed that one of theVitisAAMTs shares an ancestor with jasmonic acid methyltransferases, similar to the AAMT from strawberry (Frageriasp.). Collectively, these data demonstrate the molecular mechanisms underpinning AA activity across methyltransferases and identify one‐step enzymes by which grapes synthesize MeAA.
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The Mutational Road not Taken: Using Ancestral Sequence Resurrection to Evaluate the Evolution of Plant Enzyme Substrate Preferences
Abstract We investigated the flowering plant salicylic acid methyl transferase (SAMT) enzyme lineage to understand the evolution of substrate preference change. Previous studies indicated that a single amino acid replacement to the SAMT active site (H150M) was sufficient to change ancestral enzyme substrate preference from benzoic acid to the structurally similar substrate, salicylic acid (SA). Yet, subsequent studies have shown that the H150M function-changing replacement did not likely occur during the historical episode of enzymatic divergence studied. Therefore, we reinvestigated the origin of SA methylation preference here and additionally assessed the extent to which epistasis may act to limit mutational paths. We found that the SAMT lineage of enzymes acquired preference to methylate SA from an ancestor that preferred to methylate benzoic acid as previously reported. In contrast, we found that a different amino acid replacement, Y267Q, was sufficient to change substrate preference with others providing small positive-magnitude epistatic improvements. We show that the kinetic basis for the ancestral enzymatic change in substate preference by Y267Q appears to be due to both a reduced specificity constant, kcat/KM, for benzoic acid and an improvement in KM for SA. Therefore, this lineage of enzymes appears to have had multiple mutational paths available to achieve the same evolutionary divergence. While the reasons remain unclear for why one path was taken, and the other was not, the mutational distance between ancestral and descendant codons may be a factor.
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- Award ID(s):
- 2325341
- PAR ID:
- 10515321
- Editor(s):
- Zhang, George
- Publisher / Repository:
- Oxford Academic
- Date Published:
- Journal Name:
- Genome Biology and Evolution
- Volume:
- 16
- Issue:
- 2
- ISSN:
- 1759-6653
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1093/gbe/evae016
