The alarming rise of antimicrobial resistance requires antibiotics with unexploited mechanisms. Ideal templates could be antibiotics that target the peptidoglycan precursor lipid II, known as the bacterial Achilles heel, at an irreplaceable pyrophosphate group. Such antibiotics would kill multidrug-resistant pathogens at nanomolecular concentrations without causing antimicrobial resistance. However, due to the challenge of studying small membrane-embedded drug–receptor complexes in native conditions, the structural correlates of the pharmaceutically relevant binding modes are unknown. Here, using advanced highly sensitive solid-state NMR setups, we present a high-resolution approach to study lipid II-binding antibiotics directly in cell membranes. On the example of nisin, the preeminent lantibiotic, we show that the native antibiotic-binding mode strongly differs from previously published structures, and we demonstrate that functional hotspots correspond to plastic drug domains that are critical for the cellular adaptability of nisin. Thereby, our approach provides a foundation for an improved understanding of powerful antibiotics.

Geraniol derived from essential oils of various plant species is widely used in the cosmetic and perfume industries. It is also an essential trait of the pleasant smell of rose flowers. In contrast to other monoterpenes which are produced in plastids via the methyl erythritol phosphate pathway, geraniol biosynthesis in roses relies on cytosolic NUDX1 hydrolase which dephosphorylates geranyl diphosphate (GPP). However, the metabolic origin of cytosolic GPP remains unknown. By feeding Rosa chinensis “Old Blush” flowers with pathway-specific precursors and inhibitors, combined with metabolic profiling and functional characterization of enzymes in vitro and in planta, we show that geraniol is synthesized through the cytosolic mevalonate (MVA) pathway by a bifunctional geranyl/farnesyl diphosphate synthase, RcG/FPPS1, producing both GPP and farnesyl diphosphate (FPP). The downregulation and overexpression of RcG/FPPS1 in rose petals affected not only geraniol and germacrene D emissions but also dihydro-β-ionol, the latter due to metabolic cross talk of RcG/FPPS1-dependent isoprenoid intermediates trafficking from the cytosol to plastids. Phylogenetic analysis together with functional characterization of G/FPPS orthologs revealed that the G/FPPS activity is conserved among Rosaceae species. Site-directed mutagenesis and molecular dynamic simulations enabled to identify two conserved amino acids that evolved from ancestral FPPSs and contribute to GPP/FPP product specificity. Overall, this study elucidates the origin of the cytosolic GPP for NUDX1-dependent geraniol production, provides insights into the emergence of the RcG/FPPS1 GPPS activity from the ancestral FPPSs, and shows that RcG/FPPS1 plays a key role in the biosynthesis of volatile terpenoid compounds in rose flowers.

Plant isoprenoids are derived from two biosynthetic pathways, the cytoplasmic mevalonate (MVA) and the plastidial methylerythritol phosphate (MEP) pathway. In this study their respective contributions toward formation of dolichols in Coluria geoides hairy root culture were estimated using in vivo labeling with ¹³C-labeled glucose as a general precursor. NMR and mass spectrometry showed that both the MVA and MEP pathways were the sources of isopentenyl diphosphate incorporated into polyisoprenoid chains. The involvement of the MEP pathway was found to be substantial at the initiation stage of dolichol chain synthesis, but it was virtually nil at the terminal steps; statistically, 6–8 isoprene units within the dolichol molecule (i.e. 40–50% of the total) were derived from the MEP pathway. These results were further verified by incorporation of [5-²H]mevalonate or [5,5-²H₂]deoxyxylulose into dolichols as well as by the observed decreased accumulation of dolichols upon treatment with mevinolin or fosmidomycin, selective inhibitors of either pathway. The presented data indicate that the synthesis of dolichols in C. geoides roots involves a continuous exchange of intermediates between the MVA and MEP pathways. According to our model, oligoprenyl diphosphate chains of a length not exceeding 13 isoprene units are synthesized in plastids from isopentenyl diphosphate derived from both the MEP and MVA pathways, and then are completed in the cytoplasm with several units derived solely from the MVA pathway. This study also illustrates an innovative application of mass spectrometry for qualitative and quantitative evaluation of the contribution of individual metabolic pathways to the biosynthesis of natural products.

A plastidial cis-prenyltransferase synthesizes a family of polyprenols that accumulate in thylakoid membranes and play a central role in the efficiency of photo