Molecular Ecology and Evolution
Barkman, Todd J. , Martins, Talline , Sutton, Elizabeth , Stout, John .
Positive selection for single amino acid change promotes substrate discrimination of a plant volatile-producing enzyme.
We used a combined evolutionary and experimental approach to better understand enzyme functional divergence within the SABATH gene family of methyltransferases. These enzymes catalyze the formation of a variety of secondary metabolites in plants, many of which are volatiles that contribute to floral scent and plant defense such as methyl salicylate and methyl jasmonate. A phylogenetic analysis of functionally characterized members of this family showed that salicylic acid methyltransferase (SAMT) forms a monophyletic lineage of sequences found in several flowering plants. Most members of this lineage preferentially methylate salicylic acid as compared to the structurally similar substrate benzoic acid. To investigate if positive selection promoted functional divergence of this lineage of enzymes, we performed a branch-sites test. This test showed statistically-significant support for positive selection. A high posterior probability identified an active site methionine as the only site under positive selection in this lineage. To investigate the potential catalytic effect of this positively-selected codon, site-directed mutagenesis was used to replace Met with the alternative amino acid (His) in a Datura wrightii floral-expressed SAMT sequence. Heterologous expression of wild-type and mutant D. wrightii SAMT in E. coli showed that both enzymes could convert salicylic acid to methyl salicylate and benzoic acid to methyl benzoate. However, competitive feeding with equimolar amounts of salicylic acid and benzoic acid showed that the presence of Met in the active site of wild-type SAMT resulted in a >10-fold higher amount of methyl salicylate produced relative to methyl benzoate. The Met156His-mutant exhibited little differential preference for the two substrates, because nearly equal amounts of methyl salicylate and methyl benzoate were produced. Evolution of the ability to discriminate between the two substrates by SAMT may be advantageous for efficient production of methyl salicylate, which is important for pollinator attraction as well as pathogen and herbivore defense.
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1 - Western Michigan University, Department of Biological Sciences, 1903 West Michigan Ave, Kalamazoo, Michigan, 49008, USA
2 - University of Wisconsin, Department of Botany, Madison, WI, 53706, USA
3 - Western Michigan University, Department of Biological Sciences, 1903 West Michigan Ave, Kalamazoo, MI, 49008, USA
Presentation Type: Oral Paper:Papers for Topics
Location: Boulevard B/Hilton
Date: Wednesday, July 11th, 2007
Time: 1:45 PM