The Mutational Road not Taken: Using Ancestral Sequence Resurrection to Evaluate the Evolution of Plant Enzyme Substrate Preferences.

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.

A Collaborative Classroom Investigation of the Evolution of SABATH Methyltransferase Substrate Preference Shifts over 120 My of Flowering Plant History.

Next-generation sequencing has resulted in an explosion of available data, much of which remains unstudied in terms of biochemical function; yet, experimental characterization of these sequences has the potential to provide unprecedented insight into the evolution of enzyme activity. One way to make inroads into the experimental study of the voluminous data available is to engage students by integrating teaching and research in a college classroom such that eventually hundreds or thousands of enzymes may be characterized. In this study, we capitalize on this potential to focus on SABATH methyltransferase enzymes that have been shown to methylate the important plant hormone, salicylic acid (SA), to form methyl salicylate. We analyze data from 76 enzymes of flowering plant species in 23 orders and 41 families to investigate how widely conserved substrate preference is for SA methyltransferase orthologs. We find a high degree of conservation of substrate preference for SA over the structurally similar metabolite, benzoic acid, with recent switches that appear to be associated with gene duplication and at least three cases of functional compensation by paralogous enzymes. The presence of Met in active site position 150 is a useful predictor of SA methylation preference in SABATH methyltransferases but enzymes with other residues in the homologous position show the same substrate preference. Although our dense and systematic sampling of SABATH enzymes across angiosperms has revealed novel insights, this is merely the "tip of the iceberg" since thousands of sequences remain uncharacterized in this enzyme family alone.