The objective of the proposed research is to understand the molecular evolution of four subfamilies of genes within the large and diverse family of genes encoding the 2-oxoglutarate-dependent dioxygenases (2-ODDs). The subfamilies of 2-ODDs include enzymes that are part of a number of fundamental biosynthetic pathways including gibberellic acid biosynthesis, flavonoid biosynthesis, and alkaloid biosynthesis in plants and beta lactam antibiotic biosynthesis in fungi. Specific aspects of molecular evolution to be considered include: 1) characterization of gene copy number and determination of orthologous and paralogous relationships among genes in two small multigene subfamilies encoding the 2-oxidases and the 20-oxidases in a group of related legume species; 2) phylogenetic analyses of relationships among four subfamilies of genes coding for 3 beta-hydroxylase, 2-oxidase, 20-oxidase, and flavanone-3-hydroxylase within the genes coding for 2-ODDs; and 3) consideration of the relationship between amino acid sequence and function of enzymes coded by genes for different subfamilies of 2-ODDs. All enzymes in the 2-ODD superfamily share a number of conserved amino acids that are functionally important, including the ligands for binding iron and the oxoglutarate co-substrate. Other sites are conserved within each of the subfamilies, but vary between them and may represent sites important for binding specific substrates (enzymes from different subfamilies utilize different substrates). Sites will be compared at each of two levels, a) within subfamilies and thus potentially specific to utilization of a specific substrate, and b) among all four subfamilies and thus more generally conserved among 2-ODDs. 4) test for positive selection among both recently and older duplicated genes and at sites important in substrate binding.
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