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

Abstract

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.

Keywords: enzyme evolution; paralog compensation; plant methyltransferase evolution; substrate preference evolution.

Fig. 1.

Representative structures recognized as substrates by the SABATH family of MT enzymes. Most characterized family members are carboxyl MTs and usually exhibit a high degree of preference for one substrate.

Fig. 2.

A phylogenetic analysis of 1,578 SABATH protein MT sequences shows overall enzyme family relationships. Enzyme names shown in bold have been demonstrated to methylate SA and/or benzoic acid (BA). Angiosperm enzymes that methylate SA and/or BA are found in the SAMT, BAMT, BSMT, and XMT clades. The Gymnosperm and Liverwort SAMT enzymes that methylate SA are not closely related to those of angiosperms. Bootstrap support is shown for selected nodes that separate the SA and BA-methylating clades of enzymes from others. SAMT, salicylic acid MT; BAMT, benzoic acid MT; BSMT, benzoic/salicylic acid MT; JMT, jasmonic acid MT; XMT, xanthine alkaloid MT; CS, caffeine synthase; IAMT, indole acetic acid MT; GA, Gibberellic acid MT; FAMT, farnesoic acid MT; LAMT, loganic acid MT.

Fig. 3.

A phylogenetic estimate of SAMT sequences shows that most orders of angiosperms possess orthologs except for monocots. Because SAMT has been retained in several basal angiosperms, basal eudicots, rosids and asterids, orthologs are predicted to have been present in ancestral angiosperms. Lineages marked in orange include at least one enzyme for which functional analyses have been performed as shown in figure 4. Nodes for orders supported by bootstrap proportions >90 are marked by filled blue circles.

Fig. 4.

A phylogenetic context for enzyme substrate preference in SAMT, BAMT, BSMT, and XMT enzymes. Horizontal bars show relative enzyme preference for SA and BA. Enzyme names marked with “*” are adapted from published literature. Phylogenetic relationships among enzymes are shown with branches colored according to estimated/observed active site amino acid residue. Although every enzyme with Met at position 150 prefers to methylate SA, those with His prefer BA or another substrate. Enzymes with Gln may show preference for one or the other substrate. Pie charts at selected nodes show probabilities for estimated ancestral amino acid state.

Fig. 5.

Average level of SA preference for enzymes with different amino acid residues in active site position 150. Phylogenetic ANOVA results indicate that enzymes with Gln and Met are not significantly different from one another. However, enzymes with His in the active site have significantly lower SA methylation preference than those with either Gln or His (P = 0.003 in both cases).