Project description:Terpenes synthases typically form complex molecular scaffolds by concerted activation and cyclization of linear starting materials in a single active site. We have determined that iridoid synthase, an atypical reductive terpene synthase, catalyses the activation of its substrate 8-oxogeranial into a reactive enol intermediate but does not catalyse the subsequent cyclisation into nepetalactol. This discovery led us to identify a class of nepetalactol-related short-chain dehydrogenase enzymes (NEPS) from catmint (Nepeta mussinii) which catalyse the stereoselective cyclisation of the enol intermediate into nepetalactol isomers. Subsequent oxidation of nepetalactols by NEPS1 provides nepetalactones, metabolites that are well known for both insect-repellent activity and euphoric effect in cats. Structural characterisation of the NEPS3 cyclase reveals it binds to NAD+ yet does not utilise it chemically for a non-oxidoreductive formal [4+2] cyclisation. These discoveries will complement metabolic reconstructions of iridoid and monoterpene indole alkaloid biosynthesis.
Project description:Wild yeast and many clinical strains form complex, biofilm colonies, providing protection from desiccation, drugs and other stresses. Few transcriptomic studies have focused on sub-surface invasive “roots” due to the challenges of cell isolation from agar and subpopulation cross-contamination. Here we present, a first transcriptomic analysis via RNA sequencing of root and aerial cells, separated by a novel method. We identified 719 coding genes with upregulated root expression, mainly involved in ribosome structure/biogenesis, biosynthesis and translation and 529 loci/genes with upregulated aerial expression, mainly involved in meiosis/sporulation, stress defense and protein degradation; all indicating that aerial cells are resting and root cells are metabolically active cells. On the basis of 172 root-upregulated and 233 aerial-upregulated non-coding loci detected, anti-regulated gene/lncRNA pairs have been identified that may contribute to negative regulation of gene expression. Importantly, cells showing typical markers of “roots” correspond with cells embedded in extracellular matrix and cells showing typical markers of “aerial” parts with ECM-free cells.
Project description:Vasculogenic mimicry has been generally accepted as a new form of tumor vascularization and regarded as an unfavorable prognostic factor in multiple aggressive malignancies. We previously reported the presence of vasculogenic mimicry in osteosarcoma patients. The mechanistic basis for osteosarcoma VM remains unclear. We used microarrays to detail the global programme of gene expression between 143B cells and HOS cells exposed to Matrigel which showed greatly different vasculogenic mimicry formation potential and identified distinct classes of vasculogenic mimicry-realted genes during this process.
Project description:Monoterpene indole alkaloids (MIAs) are a structurally diverse family of specialized metabolites mainly produced in Gentianales to cope with environmental challenges. Due to their pharmacological properties, the biosynthetic modalities of several MIA types have been elucidated but not that of the yohimbanes. Here, we combine transcriptomics and genome sequencing of Rauvolfia tetraphylla with machine learning to discover the unexpected multiple actors of this natural product's synthesis. We identify a medium chain dehydrogenase/reductase (MDR) that produces a mixture of four diastereomers of yohimbanes including the well-known yohimbine and rauwolscine. In addition to this multifunctional yohimbane synthase (YOS), an MDR synthesizing mainly heteroyohimbanes and the short chain dehydrogenase vitrosamine synthase also display a yohimbane synthase side activity. Lastly, we establish that the combination of geissoschizine synthase with at least three other MDRs also produces a yohimbane mixture thus shedding light on the complex mechanisms evolved for the synthesis of these plant bioactives.