Prenylated flavin mononucleotide (prFMN) is a flavin coenzyme that helps UbiD-family microbial enzymes catalyze the decarboxylation of α,β-unsaturated carboxylic acids. Since the UbiD-family decarboxylases are involved in important metabolic processes, such as the anaerobic catabolism of aromatic acids, bacterial ubiquinone biosynthesis, and the archaeal modified mevalonate pathway, prFMN is essential for many microorganisms. Biosynthesis of prFMN via the transfer of a dimethylallyl group to the N5 of reduced FMN and subsequent cyclization is catalyzed by a UbiX-family prenyltransferase called prFMN synthase (PFS). PFSs are unique because some accept dimethylallyl phosphate (DMAP) as the prenyl donor substrate instead of dimethylallyl diphosphate (DMAPP), which is a common donor substrate for many prenyltransferases. Structural and mutagenic analyses of PFS from the methanogenic archaeon Methanosarcina mazei were performed in the present study to elucidate the detailed mechanism underlying the unique donor substrate preference of PFSs. M. mazei PFS is DMAP-specific, but it can also accept DMAPP. The crystal structures of the enzyme in complex with FMN, both FMN and DMAP, or prFMN were solved, revealing the substrate-binding residues. Point mutations at a non-conserved residue, Thr163, near the substrate-binding site changed the donor substrate specificity, primarily affecting the catalytic rate rather than substrate recognition. The T163F mutant significantly decreased its activity toward DMAPP, becoming more specific to DMAP, whereas the T163Q mutant was completely inactive when DMAPP was used for the reaction. This study provides a deeper understanding of how PFS recognizes its substrates and synthesizes prFMN, emphasizing the importance of DMAP, an overlooked metabolite.
Sou Fukuhara, Hideaki Unno, Soma Ishimine et al.· The FEBS Journal· 0 citations
Monoterpenes (C10) are a large group of specialized metabolites important for plant interactions with the environment. Their biosynthesis is well understood in seed plants, where geranyl diphosphate serves as the canonical substrate, but knowledge of monoterpene biosynthesis outside seed plants remains very limited. Here, we report neryl diphosphate (NPP)-derived monoterpene biosynthesis via a biosynthetic gene cluster in the liverwort Marchantia polymorpha. MpMTPSL2, a microbial-type terpene synthase, converts NPP into α-phellandrene and D-limonene in vitro. CRISPR knockout lines showed reduced production of both monoterpenes, providing direct genetic evidence for its in planta function. MpCPT5, a cis-prenyltransferase (CPT) family member identified through co-expression with MpMTPSL2, was confirmed to encode NPP synthase, as its knockout plants abolished α-phellandrene and D-limonene production. Subcellular localization analyses in protoplasts and stable transgenic plants demonstrated that both MpCPT5 and MpMTPSL2 localize to plastids, co-localizing across all cell types with markedly stronger signals in non-green plastids of oil-body cells. Consistent with this, expression of both genes under their respective promoters was nearly abolished in oil-body-deficient mutants and strongly upregulated in a gain-of-function line for oil-body formation. MpMTPSL2 and MpCPT5 are physically linked through a shared bidirectional promoter that drives their coexpression specific to oil body cells, forming a unique biosynthetic gene cluster whose coordinated expression is maintained by PRC2-mediated H3K27me3 repression. Phylogenetic analysis implies that NPP synthases in M. polymorpha and in flowering plants evolved independently from their respective long-chain CPT ancestors. These findings provide new insights into the mechanisms and evolution of monoterpene biosynthesis in non-seed plants. Significance statement Monoterpenes are a diverse group of specialized metabolites produced widely among land plants, yet our understanding of their biosynthesis outside seed plants remains limited. Here we report that in the liverwort Marchantia polymorpha, the non-canonical substrate neryl diphosphate is used for monoterpene biosynthesis. The functions of the monoterpene synthase gene MpMTPSL2 and the neryl diphosphate synthase gene MpCPT5 were demonstrated through CRISPR knockouts. These two genes are physically linked and share a bidirectional promoter. Promoter assays show both genes function in plastids within oil-body cells, revealing cell-type specificity. These findings shew new light on the mechanisms and evolution of monoterpene biosynthesis in non-seed plants.
Guo Wei, T. Kawaguchi, Facundo Romani et al.· bioRxiv· 0 citations