Key Amino Acids for Transferase Activity of GDSL Lipases.

The Gly-Asp-Ser-Leu (GDSL) motif of esterase/lipase family proteins (GELPs) generally exhibit esterase activity, whereas transferase activity is markedly preferred in several GELPs, including the Tanacetum cinerariifolium GDSL lipase TciGLIP, which is responsible for the biosynthesis of the natural insecticide, pyrethrin I. This transferase activity is due to the substrate affinity regulated by the protein structure and these features are expected to be conserved in transferase activity-exhibiting GELPs (tr-GELPs). In this study, we identified two amino acid residues, [N/R]208 and D484, in GELP sequence alignments as candidate key residues for the transferase activity of tr-GELPs by two-entropy analysis. Molecular phylogenetic analysis demonstrated that each tr-GELP is located in the clusters for non-tr-GELPs, and most GELPs conserve at least one of the two residues. These results suggest that the two conserved residues are required for the acquisition of transferase activity in the GELP family. Furthermore, substrate docking analyses using ColabFold-generated structure models of both natives and each of the two amino acids-mutated TciGLIPs also revealed numerous docking models for the proper access of substrates to the active site, indicating crucial roles of these residues of TciGLIP in its transferase activity. This is the first report on essential residues in tr-GELPs for the transferase activity.

Draft Genome of Tanacetum Coccineum: Genomic Comparison of Closely Related Tanacetum-Family Plants.

The plant Tanacetum coccineum (painted daisy) is closely related to Tanacetum cinerariifolium (pyrethrum daisy). However, T. cinerariifolium produces large amounts of pyrethrins, a class of natural insecticides, whereas T. coccineum produces much smaller amounts of these compounds. Thus, comparative genomic analysis is expected to contribute a great deal to investigating the differences in biological defense systems, including pyrethrin biosynthesis. Here, we elucidated the 9.4 Gb draft genome of T. coccineum, consisting of 2,836,647 scaffolds and 103,680 genes. Comparative analyses of the draft genome of T. coccineum and that of T. cinerariifolium, generated in our previous study, revealed distinct features of T. coccineum genes. While the T. coccineum genome contains more numerous ribosome-inactivating protein (RIP)-encoding genes, the number of higher-toxicity type-II RIP-encoding genes is larger in T. cinerariifolium. Furthermore, the number of histidine kinases encoded by the T. coccineum genome is smaller than that of T. cinerariifolium, suggesting a biological correlation with pyrethrin biosynthesis. Moreover, the flanking regions of pyrethrin biosynthesis-related genes are also distinct between these two plants. These results provide clues to the elucidation of species-specific biodefense systems, including the regulatory mechanisms underlying pyrethrin production.

Draft genome of Tanacetum cinerariifolium, the natural source of mosquito coil.

Pyrethrum (Tanacetum cinerariifolium), which is a perennial Asteraceae plant with white daisy-like flowers, is the original source of mosquito coils and is known for the biosynthesis of the pyrethrin class of natural insecticides. However, the molecular basis of the production of pyrethrins by T. cinerariifolium has yet to be fully elucidated. Here, we present the 7.1-Gb draft genome of T. cinerariifolium, consisting of 2,016,451 scaffolds and 60,080 genes predicted with high confidence. Notably, analyses of transposable elements (TEs) indicated that TEs occupy 33.84% of the genome sequence. Furthermore, TEs of the sire and oryco clades were found to be enriched in the T. cinerariifolium-specific evolutionary lineage, occupying a total of 13% of the genome sequence, a proportion approximately 8-fold higher than that in other plants. InterProScan analysis demonstrated that biodefense-related toxic proteins (e.g., ribosome inactivating proteins), signal transduction-related proteins (e.g., histidine kinases), and metabolic enzymes (e.g., lipoxygenases, acyl-CoA dehydrogenases/oxygenases, and P450s) are also highly enriched in the T. cinerariifolium genome. Molecular phylogenetic analysis detected a variety of enzymes with genus-specific multiplication, including both common enzymes and others that appear to be specific to pyrethrin biosynthesis. Together, these data identify possible novel components of the pyrethrin biosynthesis pathway and provide new insights into the unique genomic features of T. cinerariifolium.