Promiscuous Oxidosqualene Cyclases from Neoalsomitra integrifoliola Catalyzing the Formation of Tetracyclic, Pentacyclic, and Heterocyclic Triterpenes.

Six oxidosqualene cyclases (NiOSC1-NiOSC6) from Neoalsomitra integrifoliola were characterized for the biosynthesis of diverse triterpene scaffolds, including tetracyclic and pentacyclic triterpenes from the 2,3-oxidosqualene (1) and oxacyclic triterpenes from the 2,3:22,23-dioxidosqualene (2). NiOSC1 showed high efficiency in the production of naturally rare (20R)-epimers of oxacyclic triterpenes. Mutagenesis results revealed that the NiOSC1-F731G mutant significantly increased the yields of (20R)-epimers compared to the wild type. Homology modeling and molecular docking elucidated the origin of the (20R)-configuration in the epoxide addition step.

Characterization of a Structurally Distinct ATP-Dependent Reactivating Factor of Adenosylcobalamin-Dependent Lysine 5,6-Aminomutase.

Several anaerobic bacterial species, including the Gram-negative oral bacterium Fusobacterium nucleatum, ferment lysine to produce butyrate, acetate, and ammonia. The second step of the metabolic pathway─isomerization of ß-l-lysine to erythro-3,5-diaminohexanoate─is catalyzed by the adenosylcobalamin (AdoCbl) and pyridoxal 5'-phosphate (PLP)-dependent enzyme, lysine 5,6-aminomutase (5,6-LAM). Similar to other AdoCbl-dependent enzymes, 5,6-LAM undergoes mechanism-based inactivation due to loss of the AdoCbl 5'-deoxyadenosyl moiety and oxidation of the cob(II)alamin intermediate to hydroxocob(III)alamin. Herein, we identified kamB and kamC, two genes responsible for ATP-dependent reactivation of 5,6-LAM. KamB and KamC, which are encoded upstream of the genes corresponding to α and ß subunits of 5,6-LAM (kamD and kamE), co-purified following coexpression of the genes in Escherichia coli. KamBC exhibited a basal level of ATP-hydrolyzing activity that was increased 35% in a reaction mixture that facilitated 5,6-LAM turnover with ß-l-lysine or d,l-lysine. Ultraviolet-visible (UV-vis) spectroscopic studies performed under anaerobic conditions revealed that KamBC in the presence of ATP/Mg2+ increased the steady-state concentration of the cob(II)alamin intermediate in the presence of excess ß-l-lysine. Using a coupled UV-visible spectroscopic assay, we show that KamBC is able to reactivate 5,6-LAM through exchange of the damaged hydroxocob(III)alamin for AdoCbl. KamBC is also specific for 5,6-LAM as it had no effect on the rate of substrate-induced inactivation of the homologue, ornithine 4,5-aminomutase. Based on sequence homology, KamBC is structurally distinct from previously characterized B12 chaperones and reactivases, and correspondingly adds to the list of proteins that have evolved to maintain the cellular activity of B12 enzymes.

Disruption of UDP-galactopyranose mutase expression: A novel strategy for regulation of galactomannan biosynthesis and monascus pigments secretion in Monascus purpureus M9.

The impact of the cell wall structure of Monascus purpureus M9 on the secretion of extracellular monascus pigments (exMPs) was investigated. To modify the cell wall structure, UDP-galactopyranose mutase (GlfA) was knocked out using Agrobacterium-mediated transformation method, leading to a significant reduction in the Galf-based polysaccharide within the cell wall. Changes in mycelium morphology, sporogenesis, and the expression of relevant genes in M9 were also observed following the mutation. Regarding MPs secretion, a notable increase was observed in six types of exMPs (R1, R2, Y1, Y2, O1 and O2). Specifically, these exMPs exhibited enhancement of 1.33, 1.59, 0.8, 2.45, 2.89 and 4.03 times, respectively, compared to the wild-type strain. These findings suggest that the alteration of the cell wall structure could selectively influence the secretion of MPs in M9. The underlying mechanisms were also discussed. This research contributes new insights into the regulation of the synthesis and secretion of MPs in Monascus spp..

Insight into neofunctionalization of 2,3-oxidosqualene cyclases in B,C-ring-opened triterpene biosynthesis in quinoa.

Bioactive triterpenes feature complex fused-ring structures, primarily shaped by the first-committed enzyme, 2,3-oxidosqualene cyclases (OSCs) in plant triterpene biosynthesis. Triterpenes with B,C-ring-opened skeletons are extremely rare with unknown formation mechanisms, harbouring unchartered chemistry and biology. Here, through mining the genome of Chenopodium quinoa followed by functional characterization, we identified a stress-responsive and neofunctionalized OSC capable of generating B,C-ring-opened triterpenes, including camelliol A and B and the novel (-)-quinoxide A as wax components of the specialized epidermal bladder cells, namely the quinoxide synthase (CqQS). Protein structure analysis followed by site-directed mutagenesis identified key variable amino acid sites underlying functional interconversion between pentacyclic ß-amyrin synthase (CqbAS1) and B,C-ring-opened triterpene synthase CqQS. Mutation of one key residue (N612K) in even evolutionarily distant Arabidopsis ß-amyrin synthase could generate quinoxides, indicating a conserved mechanism for B,C-ring-opened triterpene formation in plants. Quantum computation combined with docking experiments further suggests that conformations of conserved W613 and F413 of CqQS might be key to selectively stabilizing intermediate carbocations towards B,C-ring-opened triterpene formation. Our findings shed light on quinoa triterpene skeletal diversity and mechanisms underlying B,C-ring-opened triterpene biosynthesis, opening avenues towards accessing their chemistry and biology and paving the way for quinoa trait engineering and quality improvement.

A case of congenital cataracts with hypotrichosis caused by compound heterozygous variants in the LSS gene.

BACKGROUND: Patients with biallelic variants in the lanosterol synthase (LSS) gene has been reported to exhibit phenotypes as follows: non-syndromic form of hypotrichosis, congenital cataracts, and alopecia with intellectual disability or growth retardation. However, genotype-phenotype correlations in the LSS gene are still not completely clear. METHODS: In this study, we reported a Chinese girl who had congenital cataracts with hypotrichosis. The trio exome sequencing was performed to elucidate the genetic cause of the patient. RESULTS: We identified compound heterozygous variants (c.296G>A, p.G99D and c.1025T>G, p.I342S) in the LSS gene. Both variants altered the amino acid coding at highly conserved amino acid residues and were predicted to be deleterious using prediction software. CONCLUSION: Our report expands the spectrum of variants in the LSS gene and will be helpful for genotype-phenotype correlations study.

Cloning and functional characterization of the oxidative squalene cyclase gene in the deep-sea holothurian Chiridota sp.

Saponins derived from holothurians have high potential medicinal value. However, the de novo synthesis of the derivatization of triterpenes is still unclear. Oxidative squalene cyclase (OSC) can catalyze 2,3-Oxidosqualene into diverse products that serve as important precursors for triterpene synthesis. However, the function of theOSCgene in Chiridotasp. hasnot been elucidated. In this study, an OSCgenederived from the deep-sea holothurianChiridota sp. was cloned and characterized functionally in a yeast system. The open reading frame of the OSC gene was 2086 bp, which encoded 695 amino acids. The Chiridota sp. OSC gene has a similarity of 66.89 % to the OSC of other holothurian species and 63.51 % to that of Acanthaster planci. The phylogenetic tree showed that the echinozoan OSCsclustered together, and then they formeda sister group to fungi and plant homologs. Chiridota sp. OSC catalyzed 2,3-Oxidosqualene into parkeol.Under high pressure, the relative enzymatic activity and stability of cyclase inChiridota sp. was higher than that in the shallow-sea holothurianStichopus horrens. The newly cloned OSC of Chiridota sp.provideskey information for the interpretation of the saponin synthesis pathway in deep-sea holothurians.

Expanding Pseudomonas taiwanensis VLB120's acyl-CoA portfolio: Propionate production in mineral salt medium.

As one of the main precursors, acetyl-CoA leads to the predominant production of even-chain products. From an industrial biotechnology perspective, extending the acyl-CoA portfolio of a cell factory is vital to producing industrial relevant odd-chain alcohols, acids, ketones and polyketides. The bioproduction of odd-chain molecules can be facilitated by incorporating propionyl-CoA into the metabolic network. The shortest pathway for propionyl-CoA production, which relies on succinyl-CoA catabolism encoded by the sleeping beauty mutase operon, was evaluated in Pseudomonas taiwanensis VLB120. A single genomic copy of the sleeping beauty mutase genes scpA, argK and scpB combined with the deletion of the methylcitrate synthase PVLB_08385 was sufficient to observe propionyl-CoA accumulation in this Pseudomonas. The chassis' capability for odd-chain product synthesis was assessed by expressing an acyl-CoA hydrolase, which enabled propionate synthesis. Three fed-batch strategies during bioreactor fermentations were benchmarked for propionate production, in which a maximal propionate titre of 2.8 g L-1 was achieved. Considering that the fermentations were carried out in mineral salt medium under aerobic conditions and that a single genome copy drove propionyl-CoA production, this result highlights the potential of Pseudomonas to produce propionyl-CoA derived, odd-chain products.

OXIDOSQUALENE CYCLASE 1 and 2 influence triterpene biosynthesis and defense in Nicotiana attenuata.

Triterpenes are a class of bioactive compounds with diverse biological functions, playing pivotal roles in plant defense against biotic stressors. Oxidosqualene cyclases (OSCs) serve as gatekeepers in the biosynthesis of triterpenes. In this study, we utilized a Nicotiana benthamiana heterologous expression system to characterize NaOSC1 from Nicotiana attenuata as a multifunctional enzyme capable of synthesizing lupeol, dammarenediol II, 3-alpha,20-lupanediol, and 7 other triterpene scaffolds. We also demonstrated that NaOSC2 is, in contrast, a selective enzyme, producing only the ß-amyrin scaffold. Through virus-induced gene silencing and in vitro toxicity assays, we elucidated the roles of NaOSC1 and NaOSC2 in the defense of N. attenuata against Manduca sexta larvae. Metabolomic and feature-based molecular network analyses of leaves with silenced NaOSC1 and NaOSC2 unveiled 3 potential triterpene glycoside metabolite clusters. Interestingly, features identified as triterpenes within these clusters displayed a significant negative correlation with larval mass. Our study highlights the pivotal roles of NaOSC1 and NaOSC2 from N. attenuata in the initial steps of triterpene biosynthesis, subsequently influencing defense against M. sexta through the modulation of downstream triterpene glycoside compounds.

The structural basis of mRNA recognition and binding by yeast pseudouridine synthase PUS1.

The chemical modification of RNA bases represents a ubiquitous activity that spans all domains of life. Pseudouridylation is the most common RNA modification and is observed within tRNA, rRNA, ncRNA and mRNAs. Pseudouridine synthase or 'PUS' enzymes include those that rely on guide RNA molecules and others that function as 'stand-alone' enzymes. Among the latter, several have been shown to modify mRNA transcripts. Although recent studies have defined the structural requirements for RNA to act as a PUS target, the mechanisms by which PUS1 recognizes these target sequences in mRNA are not well understood. Here we describe the crystal structure of yeast PUS1 bound to an RNA target that we identified as being a hot spot for PUS1-interaction within a model mRNA at 2.4 Å resolution. The enzyme recognizes and binds both strands in a helical RNA duplex, and thus guides the RNA containing the target uridine to the active site for subsequent modification of the transcript. The study also allows us to show the divergence of related PUS1 enzymes and their corresponding RNA target specificities, and to speculate on the basis by which PUS1 binds and modifies mRNA or tRNA substrates.

Structural and Catalytic Insight into the Unique Pentacyclic Triterpene Synthase TwOSC.

The oxidosqualene cyclase (OSC) catalyzed cyclization of the linear substrate (3S)-2,3-oxidosqualene to form diverse pentacyclic triterpenoid (PT) skeletons is one of the most complex reactions in nature. Friedelin has a unique PT skeleton involving a fascinating nine-step cation shuttle run (CSR) cascade rearrangement reaction, in which the carbocation formed at C2 moves to the other side of the skeleton, runs back to C3 to yield a friedelin cation, which is finally deprotonated. However, as crystal structure data of plant OSCs are lacking, it remains unknown why the CSR cascade reactions occur in friedelin biosynthesis, as does the exact catalytic mechanism of the CSR. In this study, we determined the first cryogenic electron microscopy structure of a plant OSC, friedelin synthase, from Tripterygium wilfordii Hook. f (TwOSC). We also performed quantum mechanics/molecular mechanics simulations to reveal the energy profile for the CSR cascade reaction and identify key residues crucial for PT skeleton formation. Furthermore, we semirationally designed two TwOSC mutants, which significantly improved the yields of friedelin and ß-amyrin, respectively.

Biphosphoglycerate Mutase: A Novel Therapeutic Target for Malaria?

Biphosphoglycerate mutase (BPGM) is a tri-functional enzyme expressed exclusively in erythroid cells and tissues that is responsible for the production of 2,3-biphosphoglycerate (2,3-BPG) through the Rapoport-Luebering shunt. The 2,3-BPG is required for efficient glycolysis and ATP production under anaerobic conditions, but is also a critical allosteric regulator of hemoglobin (Hb), acting to regulate oxygen release in peripheral tissues. In humans, BPGM deficiency is very rare, and is associated with reduced levels of erythrocytic 2,3-BPG and ATP, left shifted Hb-O2 dissociation curve, low P50, elevated Hb and constitutive erythrocytosis. BPGM deficiency in mice recapitulates the erythroid defects seen in human patients. A recent report has shown that BPGM deficiency in mice affords striking protection against both severe malaria anemia and cerebral malaria. These findings are reminiscent of studies of another erythrocyte specific glycolytic enzyme, Pyruvate Kinase (PKLR), which mutational inactivation protects humans and mice against malaria through impairment of glycolysis and ATP production in erythrocytes. BPGM, and PKLR join glucose-6-phosphate dehydrogenase (G6PD) and other erythrocyte variants as modulating response to malaria. Recent studies reviewed suggest glycolysis in general, and BPGM in particular, as a novel pharmacological target for therapeutic intervention in malaria.

Structure-Guided Discovery of N5-CAIR Mutase Inhibitors.

Because purine nucleotides are essential for all life, differences between how microbes and humans metabolize purines can be exploited for the development of antimicrobial therapies. While humans biosynthesize purine nucleotides in a 10-step pathway, most microbes utilize an additional 11th enzymatic activity. The human enzyme, aminoimidazole ribonucleotide (AIR) carboxylase generates the product 4-carboxy-5-aminoimidazole ribonucleotide (CAIR) directly. Most microbes, however, require two separate enzymes, a synthetase (PurK) and a mutase (PurE), and proceed through the intermediate, N5-CAIR. Toward the development of therapeutics that target these differences, we have solved crystal structures of the N5-CAIR mutase of the human pathogens Legionella pneumophila (LpPurE) and Burkholderia cenocepacia (BcPurE) and used a structure-guided approach to identify inhibitors. Analysis of the structures reveals a highly conserved fold and active site architecture. Using this data, and three additional structures of PurE enzymes, we screened a library of FDA-approved compounds in silico and identified a set of 25 candidates for further analysis. Among these, we identified several new PurE inhibitors with micromolar IC50 values. Several of these compounds, including the α1-blocker Alfuzosin, inhibit the microbial PurE enzymes much more effectively than the human homologue. These structures and the newly described PurE inhibitors are valuable tools to aid in further studies of this enzyme and provide a foundation for the development of compounds that target differences between human and microbial purine metabolism.

A Fluorescence-Based Assay for N5 -Carboxyaminoimidazole Ribonucleotide Mutase.

The enzyme N5 -carboxylaminoinidazole ribonucleotide (N5 -CAIR) mutase is found in microbial de novo purine biosynthesis but is absent in humans making it an attractive antimicrobial target. N5 -CAIR mutase catalyzes the synthesis of carboxyaminoimidazole ribonucleotide (CAIR) from N5 -CAIR which is itself prepared from aminoimidazole ribonucleotide (AIR) by the enzyme N5 -CAIR synthetase. During our research on identifying inhibitors of N5 -CAIR mutase, we developed an innovative, fluorescence-based assay to measure the activity of this enzyme. This assay relies upon our recent serendipitous observation that AIR reversibly reacts with the compound isatin. Reaction of a fluorescently-tagged isatin with AIR resulted in a large increase in fluorescence intensity allowing a measurement of the concentration of AIR in solution. From this observation, we developed a reproducible, non-continuous assay that can replicate the known kinetic parameters of the enzyme and can readily detect a recognized inhibitor of the enzyme. This assay should find utility in screening for inhibitors targeting N5 -CAIR mutase.

Architecture of the human G-protein-methylmalonyl-CoA mutase nanoassembly for B12 delivery and repair.

G-proteins function as molecular switches to power cofactor translocation and confer fidelity in metal trafficking. The G-protein, MMAA, together with MMAB, an adenosyltransferase, orchestrate cofactor delivery and repair of B12-dependent human methylmalonyl-CoA mutase (MMUT). The mechanism by which the complex assembles and moves a >1300 Da cargo, or fails in disease, are poorly understood. Herein, we report the crystal structure of the human MMUT-MMAA nano-assembly, which reveals a dramatic 180° rotation of the B12 domain, exposing it to solvent. The complex, stabilized by MMAA wedging between two MMUT domains, leads to ordering of the switch I and III loops, revealing the molecular basis of mutase-dependent GTPase activation. The structure explains the biochemical penalties incurred by methylmalonic aciduria-causing mutations that reside at the MMAA-MMUT interfaces we identify here.

The 2-methylpropene degradation pathway in Mycobacteriaceae family strains.

Mycolicibacterium gadium IBE100 and Mycobacterium paragordonae IBE200 are aerobic, chemoorganoheterotrophic bacteria isolated from activated sludge from a wastewater treatment plant. They use 2-methylpropene (isobutene, 2-MP) as the sole source of carbon and energy. Here, we postulate a degradation pathway of 2-methylpropene derived from whole genome sequencing, differential expression analysis and peptide-mass fingerprinting. Key genes identified are coding for a 4-component soluble diiron monooxygenase with epoxidase activity, an epoxide hydrolase, and a 2-hydroxyisobutyryl-CoA mutase. In both strains, involved genes are arranged in clusters of 61.0 and 58.5 kbp, respectively, which also contain the genes coding for parts of the aerobic pathway of adenosylcobalamin synthesis. This vitamin is essential for the carbon rearrangement reaction catalysed by the mutase. These findings provide data for the identification of potential 2-methylpropene degraders.

Aging-induced pseudouridine synthase 10 impairs hematopoietic stem cells.

Aged hematopoietic stem cells (HSC) exhibit compromised reconstitution capacity and differentiation-bias towards myeloid lineage, however, the molecular mechanism behind it remains not fully understood. In this study, we observed that the expression of pseudouridine (Ψ) synthase 10 is increased in aged hematopoietic stem and progenitor cells (HSPC) and enforced protein of Ψ synthase 10 (PUS10) recapitulates the phenotype of aged HSC, which is not achieved by its Ψ synthase activity. Consistently, we observed no difference of transcribed RNA pseudouridylation profile between young and aged HSPC. No significant alteration of hematopoietic homeostasis and HSC function is observed in young Pus10-/- mice, while aged Pus10-/- mice exhibit mild alteration of hematopoietic homeostasis and HSC function. Moreover, we observed that PUS10 is ubiquitinated by E3 ubiquitin ligase CRL4DCAF1 complex and the increase of PUS10 in aged HSPC is due to aging-declined CRL4DCAF1- mediated ubiquitination degradation signaling. Taken together, this study for the first time evaluated the role of PUS10 in HSC aging and function, and provided a novel insight into HSC rejuvenation and its clinical application.

De Novo Transcriptome Sequencing of Codonopsis lanceolata for Identification of Triterpene Synthase and Triterpene Acetyltransferase.

Codonopsis lanceolata (Campanulaceae) is a perennial plant commonly known as the bonnet bellflower. This species is widely used in traditional medicine and is considered to have multiple medicinal properties. In this study, we found that shoots and roots of C. lanceolata contained various types of free triterpenes (taraxerol, ß-amyrin, α-amyrin, and friedelin) and triterpene acetates (taraxerol acetate, ß-amyrin acetate, and α-amyrin acetate). The content of triterpenes and triterpene acetates by GC analysis was higher in the shoot than in the roots. To investigate the transcriptional activity of genes involved in triterpenes and triterpene acetate biosynthesis, we performed de novo transcriptome analysis of shoots and roots of C. lanceolata by sequencing using the Illumina platform. A total of 39,523 representative transcripts were obtained. After functional annotation of the transcripts, the differential expression of genes involved in triterpene biosynthetic pathways was investigated. Generally, the transcriptional activity of unigenes in the upstream region (MVA and MEP pathway) of triterpene biosynthetic pathways was higher in shoots than in roots. Various triterpene synthases (2,3-oxidosqualene cyclase, OSC) participate to produce triterpene skeletons by the cyclization of 2,3-oxidosqualene. A total of fifteen contigs were obtained in annotated OSCs in the representative transcripts. Functional characterization of four OSC sequences by heterologous expression in yeast revealed that ClOSC1 was determined as taraxerol synthase, and ClOSC2 was a mixed-amyrin synthase producing α-amyrin and ß-amyrin. Five putative contigs of triterpene acetyltransferases showed high homology to the lettuce triterpene acetyltransferases. Conclusively, this study provides the basis of molecular information, particularly for the biosynthesis of triterpenes and triterpene acetates in C. lanceolata.

Harnessing the Structure and Dynamics of the Squalene-Hopene Cyclase for (-)-Ambroxide Production.

Terpene cyclases offer enormous synthetic potential, given their unique ability to forge complex hydrocarbon scaffolds from achiral precursors within a single cationic rearrangement cascade. Harnessing their synthetic power, however, has proved to be challenging owing to their generally low catalytic performance. In this study, we unveiled the catalytic potential of the squalene-hopene cyclase (SHC) by harnessing its structure and dynamics. First, we synergistically tailored the active site and entrance tunnel of the enzyme to generate a 397-fold improved (-)-ambroxide synthase. Our computational investigations explain how the introduced mutations work in concert to improve substrate acquisition, flow, and chaperoning. Kinetics, however, showed terpene-induced inactivation of the membrane-bound SHC to be the major turnover limitation in vivo. Merging this insight with the improved and stereoselective catalysis of the enzyme, we applied a feeding strategy to exceed 105 total turnovers. We believe that our results may bridge the gap for broader application of SHCs in synthetic chemistry.

The Identification of SQS/SQE/OSC Gene Families in Regulating the Biosynthesis of Triterpenes in Potentilla anserina.

The tuberous roots of Potentilla anserina (Pan) are an edible and medicinal resource in Qinghai-Tibetan Plateau, China. The triterpenoids from tuberous roots have shown promising anti-cancer, hepatoprotective, and anti-inflammatory properties. In this study, we carried out phylogenetic analysis of squalene synthases (SQSs), squalene epoxidases (SQEs), and oxidosqualene cyclases (OSCs) in the pathway of triterpenes. In total, 6, 26, and 20 genes of SQSs, SQEs, and OSCs were retrieved from the genome of Pan, respectively. Moreover, 6 SQSs and 25 SQEs genes expressed in two sub-genomes (A and B) of Pan. SQSs were not expanded after whole-genome duplication (WGD), and the duplicated genes were detected in SQEs. Twenty OSCs were divided into two clades of cycloartenol synthases (CASs) and ß-amyrin synthases (ß-ASs) by a phylogenetic tree, characterized with gene duplication and evolutionary divergence. We speculated that ß-ASs and CASs may participate in triterpenes synthesis. The data presented act as valuable references for future studies on the triterpene synthetic pathway of Pan.

Importance of aspartic acid side chain carboxylate-arginine interaction in substrate selection of arginine 2,3-aminomutase BlsG.

The fungicide nucleoside blasticidin S features a ß-arginine, a moiety seldom revealed in the structure of natural products. BlsG, a radical SAM arginine-2,3-aminomutase from the blasticidin S biosynthetic pathway, displayed promiscuous activity to three basic amino acids. Here in this study, we demonstrated that BlsG showed high preference toward its natural substrate arginine. The combined structural modeling, steady-state kinetics, and mutational analyses lead to the detailed understanding of the substrate recognition of BlsG. A single mutation of T340D changed the substrate preference of BlsG leading to a little more preference to lysine than arginine. On the basis of our understanding of the substrate selection of BlsG and bioinformatic analysis, we propose that the D…D motif locationally corresponding to D293 and D330 of KAM is characteristic of lysine 2,3-aminomutase while the corresponding D…T motif is characteristic of arginine 2,3-aminomutase. The study may provide a simple way to discern the arginine 2,3-aminomutase and thus lead to the discovery of new natural compounds with ß-arginine moiety.