Aug 2026· Journal of Biological Chemistry· pp.
113510
· 0 citations· 55 references
Medicine
TL;DR
This study highlights a new role for agmatine in bacterial polyamine biosynthesis, which also includes the N1-aminopropylagmatine and carboxyaminopropylagmatine pathways for spermidine production.
Abstract
Homospermidine is a structural analog of spermidine, a polyamine that is essential for growth and cell proliferation in eukaryotes. The eukaryotic enzyme deoxyhypusine synthase (DHS) can synthesize homospermidine directly from spermidine and putrescine. Nonhomologous bacterial homospermidine synthase (HSS) synthesizes homospermidine directly from two molecules of putrescine. SpeY is a homolog of DHS in bacteria that is essential for homospermidine biosynthesis in a cyanobacterium. Recently, it was shown in the bacterial hyperthermophile Thermus thermophilus, that SpeY couples two molecules of agmatine produced by arginine decarboxylase to form N1,N9-bis(guanidino)homospermidine (G44G), which is then converted to homospermidine by a ureohydrolase. We sought to determine whether synthesis of G44G was a common feature of phylogenetically diverse SpeY homologs. Herein, we demonstrate that SpeY homologs from diverse bacterial phyla, and from species encoding arginine decarboxylases from three different protein folds, with either agmatinase or agmatine iminohydrolase partners, all form G44G. Diverse agmatine, N1-aminopropylagmatine and presumed G44G ureohydrolases convert G44G to homospermidine but SpeY-associated G44G ureohydrolases do not act on agmatine. Similarly, diverse agmatine, N1-aminopropylagmatine and presumed G44G iminohydrolases convert G44G to N1,N9-bis(carbamoyl)homospermidine but SpeY-associated G44G iminohydrolases do not act on agmatine. We identified ornithine decarboxylases in both the SpeY and HSS pathways that have become neofunctionalized to arginine decarboxylases, and in the HSS pathway are associated with an agmatinase. Bacterial homospermidine biosynthetic pathways are therefore dependent either on agmatine/SpeY or putrescine/HSS. Our study highlights a new role for agmatine in bacterial polyamine biosynthesis, which also includes the N1-aminopropylagmatine and carboxyaminopropylagmatine pathways for spermidine production.
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
Ribosomally synthesized and post-translationally modified peptides (RiPPs) are produced by biosynthetic enzymes that modify genetically encoded precursor peptide backbones and side chains. Genome mining and bioinformatics analyses targeting the multinuclear nonheme iron oxidative (MNIO) enzyme family led to the identification of a RiPP biosynthetic gene cluster from Streptomyces thermodiastaticus JCM 4840, the std cluster, which includes multiple biosynthetic enzymes and a precursor peptide containing a conserved SNKEWQE motif. Using in vitro approaches, we elucidated the modifications installed by the std biosynthetic enzymes. First, a YcaO-TfuA pair thioamidates the asparagine backbone. Next, a peptidase with an S8/S53 domain fused to a NodU-like carbamoyltransferase both carbamoylates the ε-amino group of lysine to produce the non-proteinogenic amino acid homocitrulline and cleaves the C-terminal EWQE motif. Finally, a partner protein-MNIO pair bis-hydroxylates the β- and γ-carbon positions of the installed homocitrulline to create dihydroxyhomocitrulline. The formation of homocitrulline and dihydroxyhomocitrulline is unprecedented in RiPP biosynthesis. Moreover, these findings expand the known substrate scope of YcaO-TfuA enzymes and MNIOs and identify new roles for carbamoyltransferases in these pathways.
Dayna P. Hebron, Tucker J. Shriver, Joshua J. Ziarek et al.· Journal of the American Chem...· 0 citations
Abstract Fungal iterative type I polyketide synthases (iPKSs) generate structurally diverse natural products. A subset of these assembly line enzymes, exemplified by LovB from Aspergillus terreus, contains a C-terminal condensation domain bearing a noncanonical HRxxxDG motif. While these LovB-like iPKSs are widely distributed in fungi, the majority remain uncharacterized, leaving both their associated polyketide products and the function of their unusual condensation domains largely unexplored. Here, we report the characterization of a LovB-like iPKS from Aspergillus clavatus. Heterologous reconstitution of this PKS system in Aspergillus nidulans led to the discovery of clavaic acid, a previously undescribed polyketide featuring a trimethylated trans-decalin core and an all-E-configured carboxytriene side chain. Functional analysis of the condensation domain in vivo demonstrated that it is essential for clavaic acid biosynthesis. Surprisingly, whereas the conserved arginine residue within the HRxxxDG motif was dispensable for product formation, the conserved aspartate residue was strictly required. These findings expand our understanding of LovB-like iPKSs and establish this enzyme family as a promising source of cryptic fungal polyketides. One-sentence summary Genome-guided discovery of a trans-decalin-containing polyketide encoded by a LovB-like polyketide synthase in Aspergillus clavatus.
Ping Yu, Zainab Batool, Zhen Fan et al.· Journal of Industrial Microb...· 0 citations
Iron is widely considered the first metallocofactor, evolving as iron-sulfur clusters in early life. While iron-chelating siderophores have been widely characterized across microbial life, the lanthanide-chelating metallophore, methylolanthanin, has only recently been described in Methylobacterium extorquens AM1. Methylolanthanin shares structural similarities to the siderophore rhodopetrobactin but contains 4-hydroxybenzoate chelating moieties in place of canonical 3,4-dihydroxybenzoates. Here we compare M. extorquens AM1, which produces methylolanthanin, and the closely related M. extorquens PA1, which produces rhodopetrobactin. We present a pathway for the biosynthesis of both metallophores and describe the unusual synthesis of methylolanthanin’s 4-HB moieties from tyrosine. We uncover a frameshift mutation in the predicted 3-dehydroshikimate dehydratase, mllF, that prevents production of rhodopetrobactin in AM1 through truncation of the catalytically essential N-terminus. We find that deletion of the uncharacterized gene mllG reveals a cryptic branch of the pathway, leading to production of both methylolanthanin and rhodopetrobactin. Finally, we discover that rhodopetrobactin production in this mutant is enabled through the activity of a 3-dehydroshikimate dehydratase in a separate biosynthetic gene cluster. These insights highlight an evolutionary mechanism for metallophore diversification through pseudogenization and regulation of distinct biosynthetic gene clusters with shared aromatic intermediates.
Alexa M. Zytnick, Marquis T. Yazzie, Tashi C. E. Liebergesell et al.· bioRxiv· 0 citations
Phosphoribulokinase (PRK) catalyzes the ATP-dependent phosphorylation of ribulose 5-phosphate (Ru5P) to produce ribulose 1,5-bisphosphate in the Calvin-Benson-Bassham cycle. Of PRK's four classifications, class III consists of PRKs from pseudomonadota and α-cyanobacteria. They are known as octameric enzymes regulated allosterically by NADH. In this study, we characterized the PRK from a hydrogen- and sulfur-oxidizing chemolithoautotroph Hydrogenovibrio marinus MH-110 (HmPRK). Phylogenetic analysis revealed that PRKs from organisms of Piscirickettsiaceae, including H. marinus, formed a clearly separate clade within class III. HmPRK lacks most of the conserved arginine residues which are involved in regulation by NADH. Analysis of the PRK activity in the cell-free extract of H. marinus and the purified recombinant HmPRK (rHmPRK) expressed in Escherichia coli revealed the activity as unaffected by NADH. The molecular mass of rHmPRK estimated from size-exclusion chromatography was ca. 75.4 kDa, suggesting it might form a dimer. The optimum temperature and pH of rHmPRK were 30 °C and 7.4, respectively. The enzyme had apparent Km (Ru5P) of 495 ± 108 μM and apparent Km (ATP) of 526 ± 253 μM; apparent Vmax of 298 ± 22 μmol ATP consumed min-1 mg protein-1. Regarding metabolite regulation, the enzyme activity was inhibited by phosphoenolpyruvate and slightly by AMP. These findings indicate that HmPRK has unique properties distinct from those of the canonical class III PRKs.
Tomotaka Jitsukawa, Toya Tamura, Tetsuya Ohtaki et al.· Biochemical and Biophysical...· 0 citations
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
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