Terpene cyclases catalyze the synthesis of cyclic terpenes with 10-, 15-, and 20-carbon acyclic isoprenoid diphosphates as substrates. Plants have been a source of these natural products by providing a homologous set of terpene synthases. The crystal structures of 5-epi-aristolochene synthase, a sesquiterpene cyclase from tobacco, alone and complexed separately with two farnesyl diphosphate analogs were analyzed. These structures reveal an unexpected enzymatic mechanism for the synthesis of the bicyclic product, 5-epi-aristolochene, and provide a basis for understanding the stereochemical selectivity displayed by other cyclases in the biosynthesis of pharmacologically important cyclic terpenes. As such, these structures provide templates for the engineering of novel terpene cyclases.

Galanthamine is an Amaryllidaceae alkaloid used to treat the symptoms of Alzheimer’s disease. This compound is primarily isolated from daffodil (Narcissus spp.), snowdrop (Galanthus spp.), and summer snowflake (Leucojum aestivum). Despite its importance as a medicine, no genes involved in the biosynthetic pathway of galanthamine have been identified. This absence of genetic information on biosynthetic pathways is a limiting factor in the development of synthetic biology platforms for many important botanical medicines. The paucity of information is largely due to the limitations of traditional methods for finding biochemical pathway enzymes and genes in non-model organisms. A new bioinformatic approach using several recent technological improvements was applied to search for genes in the proposed galanthamine biosynthetic pathway, first targeting methyltransferases due to strong signature amino acid sequences in the proteins. Using Illumina sequencing, a de novo transcriptome assembly was constructed for daffodil. BLAST was used to identify sequences that contain signatures for plant O-methyltransferases in this transcriptome. The program HAYSTACK was then used to identify methyltransferases that fit a model for galanthamine biosynthesis in leaf, bulb and inflorescence tissues. One candidate gene for the methylation of norbelladine to 4′-O-methylnorbelladine in the proposed galanthamine biosynthetic pathway was identified. This methyltransferase cDNA was expressed in E. coli and the protein purified by affinity chromatography. The resulting protein was found to be a norbelladine 4′-O-methyltransferase (NpN4OMT) of the proposed galanthamine biosynthetic pathway.

Trichodermamides A (1) and B (2), two modified dipeptides, have been isolated from cultures of the marine-derived fungus Trichoderma virens. The trichodermamides possess a rare cyclic O-alkyl-oxime functionality incorporated into a six-membered ring. The structure of trichodermamide B was established by X-ray diffraction analysis, while the structure assignment of trichodermamide A, and determination of the absolute stereochemistry, was accomplished by spectral and chemical methods. Trichodermamide B displayed significant in vitro cytotoxicity against HCT-116 human colon carcinoma with an IC(50) of 0.32 microg/mL.