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MBE Advance Access published online on March 20, 2007
Molecular Biology and Evolution, doi:10.1093/molbev/msm053
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© The Author 2007. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Research Article

Positive Selection for Single Amino Acid Change Promotes Substrate Discrimination of a Plant Volatile-Producing Enzyme

Todd J. Barkman, Talline Martins1, Elizabeth Sutton and John Stout

Department of Biological Sciences, Western Michigan University, Kalamazoo, MI 49008
1 Current address: Department of Botany, University of Wisconsin, Madison, WI 53706

Corresponding author: T. J. Barkman, Department of Biological Sciences, Western Michigan University, Kalamazoo, MI 49008; Phone: 269-387-2776; FAX: 269-387-5609; todd.barkman{at}wmich.edu.

Received for publication January 9, 2007. Revision received March 9, 2007. Accepted for publication March 15, 2007.

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 (p < 0.05) for positive selection in this lineage of methyltransferases (dN/dS = 10.8). A high posterior probability (pp = 0.99) 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. Since benzoic acid is a likely precursor for the biosynthesis of salicylic acid, SAMT might increase methyl salicylate levels directly by preferential methylation and indirectly by leaving more benzoic acid to be converted into salicylic acid.

Key Words: positive selection • enzyme evolution • SAMT • methyl salicylate


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