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 salicylic acid methyltransferase (SAMT) 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.

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

“…Consistent with this prediction is the recent report of conserved SAMT enzyme preference for SA, over the structurally similar BA ( Fig. 1A ), throughout angiosperm history across nearly every lineage including 41 families ( Dubs et al 2022 ). A crystal structure for Clarkia breweri SAMT was determined in 2003 ( Zubieta et al 2003 ) and revealed the importance of several residues for SA positioning for methylation in the active site including a Met150–Met308 “clamp” ( Fig.…”

“…2A , supplementary fig. S1, Supplementary Material online; adapted from Dubs et al (2022) ). Within the enzyme family, the SAMT clade is most closely related to BAMT- and XMT-type enzymes ( Dubs et al 2022 ).…”

“…No fewer than 5 published studies have used mutagenesis in modern-day and ancestral proteins to show the importance of Met150 for SA preference in different genetic backgrounds ( Zubieta et al 2003 ; Barkman et al 2007 ; Kapteyn et al 2007 ; Huang et al 2012 ; Han et al 2018 ). Yet, recently it has been shown that M150 did not evolve until much later in angiosperm history and was not likely concomitant with the origin of SA methylation preference in this clade ( Dubs et al 2022 ). Huang et al (2012) appear to have been misled by the small number of SAMT sequences available for study at that time, all of which had Met150; it is clear now that those lineages independently gained Met150 by convergence no less than 4 times in angiosperm history ( Dubs et al 2022 ).…”

“…Yet, recently it has been shown that M150 did not evolve until much later in angiosperm history and was not likely concomitant with the origin of SA methylation preference in this clade ( Dubs et al 2022 ). Huang et al (2012) appear to have been misled by the small number of SAMT sequences available for study at that time, all of which had Met150; it is clear now that those lineages independently gained Met150 by convergence no less than 4 times in angiosperm history ( Dubs et al 2022 ). Thus, it remains unclear what the mutational/structural basis for the evolution of SA preference is in this lineage of conserved enzymes.…”

Yerba mate (Ilex paraguariensis) genome provides new insights into convergent evolution of caffeine biosynthesis

“…Many nearly ubiquitous specialized metabolites involved in defence, development and floral scent are produced by SABATH enzyme family members that appear to be conserved across diverse angiosperm lineages, such as SAMT that methylates salicylic acid 55 and IAMT that methylates Indole-3-acetic acid 52 . However, caffeine is sporadically distributed amongst disparate angiosperm lineages and seems to have only recently evolved by convergence in a few distantly related orders 19 .…”