Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 109, Issue 8, Pages 2966-2971Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1019605109
Keywords
carboxyl methyltransferase; adaptive protein evolution
Categories
Funding
- National Science Foundation [DEB 0344496, MCB-1120624]
- Western Michigan University
- Deutsche Forschungsgemeinschaft [Pi 153/22]
- Direct For Biological Sciences
- Div Of Molecular and Cellular Bioscience [1120624] Funding Source: National Science Foundation
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In this study, we investigated the role for ancestral functional variation that may be selected upon to generate protein functional shifts using ancestral protein resurrection, statistical tests for positive selection, forward and reverse evolutionary genetics, and enzyme functional assays. Data are presented for three instances of protein functional change in the salicylic acid/benzoic acid/theobromine (SABATH) lineage of plant secondary metabolite-producing enzymes. In each case, we demonstrate that ancestral nonpreferred activities were improved upon in a daughter enzyme after gene duplication, and that these functional shifts were likely coincident with positive selection. Both forward and reverse mutagenesis studies validate the impact of one or a few sites toward increasing activity with ancestrally nonpreferred substrates. In one case, we document the occurrence of an evolutionary reversal of an active site residue that reversed enzyme properties. Furthermore, these studies show that functionally important amino acid replacements result in substrate discrimination as reflected in evolutionary changes in the specificity constant (k(cat)/K-M) for competing substrates, even though adaptive substitutions may affect K-M and k(cat) separately. In total, these results indicate that nonpreferred, or even latent, ancestral protein activities may be coopted at later times to become the primary or preferred protein activities.
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