Evolution of DIMBOA-Glc O-Methyltransferases from Flavonoid O-Methyltransferases in the Grasses.

Christiane Förster,Tobias G Köllner,Jonathan Gershenzon

doi:10.3390/molecules27031007

Christiane Förster, Tobias G Köllner + Show 1 more

Open Access

https://doi.org/10.3390/molecules27031007

Copy DOI

Abstract

O-Methylated benzoxazinoids (BXs) and flavonoids are widespread defenses against herbivores and pathogens in the grasses (Poaceae). Recently, two flavonoid O-methyltransferases (FOMTs), ZmFOMT2 and ZmFOMT3, have been reported to produce phytoalexins in maize (Zea mays). ZmFOMT2 and ZmFOMT3 are closely related to the BX O-methyltransferases (OMTs) ZmBX10-12 and ZmBX14, suggesting a common evolutionary origin in the Poaceae. Here, we studied the evolution and enzymatic requirements of flavonoid and BX O-methylation activities in more detail. Using BLAST searches and phylogenetic analyses, we identified enzymes homologous to ZmFOMT2 and ZmFOMT3, ZmBX10-12, and ZmBX14 in several grasses, with the most closely related candidates found almost exclusively in species of the Panicoideae subfamily. Biochemical characterization of candidate enzymes from sorghum (Sorghum bicolor), sugar cane (Saccharum spp.), and teosinte (Zea nicaraguensis) revealed either flavonoid 5-O-methylation activity or DIMBOA-Glc 4-O-methylation activity. However, DIMBOA-Glc 4-OMTs from maize and teosinte also accepted flavonols as substrates and converted them to 3-O-methylated derivatives, suggesting an evolutionary relationship between these two activities. Homology modeling, sequence comparisons, and site-directed mutagenesis led to the identification of active site residues crucial for FOMT and BX OMT activity. However, the full conversion of ZmFOMT2 activity into BX OMT activity by switching these residues was not successful. Only trace O-methylation of BXs was observed, indicating that amino acids outside the active site cavity are also involved in determining the different substrate specificities. Altogether, the results of our study suggest that BX OMTs have evolved from the ubiquitous FOMTs in the PACMAD clade of the grasses through a complex series of amino acid changes.

Highlights

O-Methylation of plant specialized metabolites is a widespread biochemical transformation that creates structural diversity and forms products with modified chemical properties that help plants cope with different biotic and abiotic stresses
Our results suggest that DIMBOA-Glc OMTs evolved from flavonoid O-methyltransferases (FOMTs) in the PACMAD clade of the grasses and that this change in activity was due to a complex series of amino acid mutations
We identified two flavonoid 5-OMTs in maize, ZmFOMT2 and ZmFOMT3, and showed that they are closely related to the DIMBOA-Glc 4-OMTs ZmBX10-12 and ZmBX14, indicating a common OMT ancestor in the grasses [28]

Summary

Introduction

O-Methylation of plant specialized metabolites is a widespread biochemical transformation that creates structural diversity and forms products with modified chemical properties that help plants cope with different biotic and abiotic stresses This tailoring reaction is catalyzed by S-adenosyl-L-methionine (SAM)-dependent O-methyltransferases (OMTs), which transfer the methyl group of the cosubstrate SAM to a hydroxyl moiety of various acceptor molecules. Plant OMTs have been classified into three major groups based on phylogenetic analysis, conserved amino acid sequence features, protein structures, and substrate specificities: caffeic acid OMTs (COMTs), caffeoyl-CoA OMTs (CCoAOMTs), and carboxylic acid OMTs [5,6,7] The latter share only marginal sequence similarities with the other classes of OMTs and are involved, for instance, in the methylation of various organic acids, such as salicylic acid, jasmonic acid, or indole-3-acetic acid [8]. Substrate specificity or regiospecificity has been associated with a single amino acid change [15,16]

Methods

Results

Conclusion