Pseudouridylation of tRNA-Derived Fragments Steers Translational Control in Stem Cells.

Pseudouridylation (Ψ) is the most abundant and widespread type of RNA epigenetic modification in living organisms; however, the biological role of Ψ remains poorly understood. Here, we show that a Ψ-driven posttranscriptional program steers translation control to impact stem cell commitment during early embryogenesis. Mechanistically, the Ψ "writer" PUS7 modifies and activates a novel network of tRNA-derived small fragments (tRFs) targeting the translation initiation complex. PUS7 inactivation in embryonic stem cells impairs tRF-mediated translation regulation, leading to increased protein biosynthesis and defective germ layer specification. Remarkably, dysregulation of this posttranscriptional regulatory circuitry impairs hematopoietic stem cell commitment and is common to aggressive subtypes of human myelodysplastic syndromes. Our findings unveil a critical function of Ψ in directing translation control in stem cells with important implications for development and disease.

Pseudouridine synthases modify human pre-mRNA co-transcriptionally and affect pre-mRNA processing.

Pseudouridine is a modified nucleotide that is prevalent in human mRNAs and is dynamically regulated. Here, we investigate when in their life cycle mRNAs become pseudouridylated to illuminate the potential regulatory functions of endogenous mRNA pseudouridylation. Using single-nucleotide resolution pseudouridine profiling on chromatin-associated RNA from human cells, we identified pseudouridines in nascent pre-mRNA at locations associated with alternatively spliced regions, enriched near splice sites, and overlapping hundreds of binding sites for RNA-binding proteins. In vitro splicing assays establish a direct effect of individual endogenous pre-mRNA pseudouridines on splicing efficiency. We validate hundreds of pre-mRNA sites as direct targets of distinct pseudouridine synthases and show that PUS1, PUS7, and RPUSD4-three pre-mRNA-modifying pseudouridine synthases with tissue-specific expression-control widespread changes in alternative pre-mRNA splicing and 3' end processing. Our results establish a vast potential for cotranscriptional pre-mRNA pseudouridylation to regulate human gene expression via alternative pre-mRNA processing.

Regulation and Function of RNA Pseudouridylation in Human Cells.

Recent advances in pseudouridine detection reveal a complex pseudouridine landscape that includes messenger RNA and diverse classes of noncoding RNA in human cells. The known molecular functions of pseudouridine, which include stabilizing RNA conformations and destabilizing interactions with varied RNA-binding proteins, suggest that RNA pseudouridylation could have widespread effects on RNA metabolism and gene expression. Here, we emphasize how much remains to be learned about the RNA targets of human pseudouridine synthases, their basis for recognizing distinct RNA sequences, and the mechanisms responsible for regulated RNA pseudouridylation. We also examine the roles of noncoding RNA pseudouridylation in splicing and translation and point out the potential effects of mRNA pseudouridylation on protein production, including in the context of therapeutic mRNAs.

Landscape of RNA pseudouridylation in archaeon Sulfolobus islandicus.

Pseudouridine, one of the most abundant RNA modifications, is synthesized by stand-alone or RNA-guided pseudouridine synthases. Here, we comprehensively mapped pseudouridines in rRNAs, tRNAs and small RNAs in the archaeon Sulfolobus islandicus and identified Cbf5-associated H/ACA RNAs. Through genetic deletion and in vitro modification assays, we determined the responsible enzymes for these modifications. The pseudouridylation machinery in S. islandicus consists of the stand-alone enzymes aPus7 and aPus10, and six H/ACA RNA-guided enzymes that account for all identified pseudouridines. These H/ACA RNAs guide the modification of all eleven sites in rRNAs, two sites in tRNAs, and two sites in CRISPR RNAs. One H/ACA RNA shows exceptional versatility by targeting eight different sites. aPus7 and aPus10 are responsible for modifying positions 13, 54 and 55 in tRNAs. We identified four atypical H/ACA RNAs that lack the lower stem and the ACA motif and confirmed their function both in vivo and in vitro. Intriguingly, atypical H/ACA RNAs can be modified by Cbf5 in a guide-independent manner. Our data provide the first global view of pseudouridylation in archaea and reveal unexpected structures, substrates, and activities of archaeal H/ACA RNPs.

Multi-omics analyses of sid2 mutant reflect the need of isochorismate synthase ICS1 to cope with sulfur limitation in Arabidopsis thaliana.

The SID2 (SA INDUCTION-DEFICIENT2) gene that encodes ICS1 (isochorismate synthase), plays a central role in salicylic acid biosynthesis in Arabidopsis. The sid2 and NahG (encoding a bacterial SA hydroxylase) overexpressing mutants (NahG-OE) have currently been shown to outperform wild type, presenting delayed leaf senescence, higher plant biomass and better seed yield. When grown under sulfate-limited conditions (low-S), sid2 mutants exhibited early leaf yellowing compared to the NahG-OE, the npr1 mutant affected in SA signaling pathway, and WT. This indicated that the hypersensitivity of sid2 to sulfate limitation was independent of the canonical npr1 SA-signaling pathway. Transcriptomic and proteomic analyses revealed that major changes occurred in sid2 when cultivated under low-S, changes that were in good accordance with early senescence phenotype and showed the exacerbation of stress responses. The sid2 mutants displayed a lower sulfate uptake capacity when cultivated under low-S and lower S concentrations in their rosettes. Higher glutathione concentrations in sid2 rosettes under low-S were in good accordance with the higher abundance of proteins involved in glutathione and ascorbate redox metabolism. Amino acid and lipid metabolisms were also strongly modified in sid2 under low-S. Depletion of total fatty acids in sid2 under low-S was consistent with the fact that S-metabolism plays a central role in lipid synthesis. Altogether, our results show that functional ICS1 is important for plants to cope with S limiting conditions.

Chromosome-level genome assembly of Prunella vulgaris L. provides insights into pentacyclic triterpenoid biosynthesis.

Prunella vulgaris is one of the bestselling and widely used medicinal herbs. It is recorded as an ace medicine for cleansing and protecting the liver in Chinese Pharmacopoeia and has been used as the main constitutions of many herbal tea formulas in China for centuries. It is also a traditional folk medicine in Europe and other countries of Asia. Pentacyclic triterpenoids are a major class of bioactive compounds produced in P. vulgaris. However, their biosynthetic mechanism remains to be elucidated. Here, we report a chromosome-level reference genome of P. vulgaris using an approach combining Illumina, ONT, and Hi-C technologies. It is 671.95 Mb in size with a scaffold N50 of 49.10 Mb and a complete BUSCO of 98.45%. About 98.31% of the sequence was anchored into 14 pseudochromosomes. Comparative genome analysis revealed a recent WGD in P. vulgaris. Genome-wide analysis identified 35 932 protein-coding genes (PCGs), of which 59 encode enzymes involved in 2,3-oxidosqualene biosynthesis. In addition, 10 PvOSC, 358 PvCYP, and 177 PvUGT genes were identified, of which five PvOSCs, 25 PvCYPs, and 9 PvUGTs were predicted to be involved in the biosynthesis of pentacyclic triterpenoids. Biochemical activity assay of PvOSC2, PvOSC4, and PvOSC6 recombinant proteins showed that they were mixed amyrin synthase (MAS), lupeol synthase (LUS), and ß-amyrin synthase (BAS), respectively. The results provide a solid foundation for further elucidating the biosynthetic mechanism of pentacyclic triterpenoids in P. vulgaris.

The galactose metabolism genes UGE1 and UGM1 are novel virulence factors of the maize anthracnose fungus Colletotrichum graminicola.

Fungal cell walls represent the frontline contact with the host and play a prime role in pathogenesis. While the roles of the cell wall polymers like chitin and branched ß-glucan are well understood in vegetative and pathogenic development, that of the most prominent galactose-containing polymers galactosaminogalactan and fungal-type galactomannan is unknown in plant pathogenic fungi. Mining the genome of the maize pathogen Colletotrichum graminicola identified the single-copy key galactose metabolism genes UGE1 and UGM1, encoding a UDP-glucose-4-epimerase and UDP-galactopyranose mutase, respectively. UGE1 is thought to be required for biosynthesis of both polymers, whereas UGM1 is specifically required for fungal-type galactomannan formation. Promoter:eGFP fusion strains revealed that both genes are expressed in vegetative and in pathogenic hyphae at all stages of pathogenesis. Targeted deletion of UGE1 and UGM1, and fluorescence-labeling of galactosaminogalactan and fungal-type galactomannan confirmed that Δuge1 mutants were unable to synthesize either of these polymers, and Δugm1 mutants did not exhibit fungal-type galactomannan. Appressoria of Δuge1, but not of Δugm1 mutants, were defective in adhesion, highlighting a function of galactosaminogalactan in the establishment of these infection cells on hydrophobic surfaces. Both Δuge1 and Δugm1 mutants showed cell wall defects in older vegetative hyphae and severely reduced appressorial penetration competence. On intact leaves of Zea mays, both mutants showed strongly reduced disease symptom severity, indicating that UGE1 and UGM1 represent novel virulence factors of C. graminicola.

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.

Glutamate 1-semialdehyde aminotransferase is connected to GluTR by GluTR-binding protein and contributes to the rate-limiting step of 5-aminolevulinic acid synthesis.

Tetrapyrroles play fundamental roles in crucial processes including photosynthesis, respiration, and catalysis. In plants, 5-aminolevulinic acid (ALA) is the common precursor of tetrapyrroles. ALA is synthesized from activated glutamate by the enzymes glutamyl-tRNA reductase (GluTR) and glutamate-1-semialdehyde aminotransferase (GSAAT). ALA synthesis is recognized as the rate-limiting step in this pathway. We aimed to explore the contribution of GSAAT to the control of ALA synthesis and the formation of a protein complex with GluTR. In Arabidopsis thaliana, two genes encode GSAAT isoforms: GSA1 and GSA2. A comparison of two GSA knockout mutants with the wild-type revealed the correlation of reduced GSAAT activity and ALA-synthesizing capacity in leaves with lower chlorophyll content. Growth and green pigmentation were more severely impaired in gsa2 than in gsa1, indicating the predominant role of GSAAT2 in ALA synthesis. Interestingly, GluTR accumulated to higher levels in gsa2 than in the wild-type and was mainly associated with the plastid membrane. We propose that the GSAAT content modulates the amount of soluble GluTR available for ALA synthesis. Several different biochemical approaches revealed the GSAAT-GluTR interaction through the assistance of GluTR-binding protein (GBP). A modeled structure of the tripartite protein complex indicated that GBP mediates the stable association of GluTR and GSAAT for adequate ALA synthesis.

A conserved mechanism affecting hydride shifting and deprotonation in the synthesis of hopane triterpenes as compositions of wax in oat.

Triterpenoids are biologically active metabolites synthesized from a common linear precursor catalyzed by 2,3-oxidosqualene cyclases (OSCs) to form diverse triterpenoid skeletons. OSCs corresponding to many discovered triterpene alcohols in nature have not been functionally and mechanistically characterized due to the diversity of chemical structures and complexity of the cyclization mechanism. We carried out a genome-wide investigation of OSCs from Avena strigosa and discovered two triterpene synthases, namely, AsHS1 and AsHS2, using a Nicotiana benthamiana expression system. These synthases produce hopenol B and hop-17(21)-en-3ß-ol, which are components of surface wax in oat panicles and sheathes, respectively. We demonstrated that substitutions of two to three amino acid residues in AsHS1 with corresponding residues from AsHS2 allowed it to be completely converted into a hop-17(21)-en-3ß-ol synthase. AsHS2 mutants with a substitution at site 410 could synthesize hopenol B alone or mixed with a side product isomotiol. The combined quantum mechanics and molecular mechanics calculation demonstrated that the side chain size of the residue at site 410 regulated the relative orientations between the hopyl C22 cation and Phe257, leading to a difference in deprotonation positions through providing or not providing cation­π interaction between the aromatic ring of F257 and the carbocation intermediate. A similar mechanism could be applied to a hopenol B synthase from a dicotyledonous plant Aquilegia. This study provided mechanistic insight into triterpenoid synthesis and discovered key amino acid residues acting on hydride transfer and a deprotonation site to differentiate between hopane-type scaffolds in diverse plant species.

Structures of the NDP-pyranose mutase belonging to glycosyltransferase family 75 reveal residues important for Mn2+ coordination and substrate binding.

Members of glycosyltransferase family 75 (GT75) not only reversibly catalyze the autoglycosylation of a conserved arginine residue with specific NDP-sugars but also exhibit NDP-pyranose mutase activity that reversibly converts specific NDP-pyranose to NDP-furanose. The latter activity provides valuable NDP-furanosyl donors for glycosyltransferases and requires a divalent cation as a cofactor instead of FAD used by UDP-D-galactopyranose mutase. However, details of the mechanism for NDP-pyranose mutase activity are not clear. Here we report the first crystal structures of GT75 family NDP-pyranose mutases. The novel structures of GT75 member MtdL in complex with Mn2+ and GDP, GDP-D-glucopyranose, GDP-L-fucopyranose, GDP-L-fucofuranose, respectively, combined with site-directed mutagenesis studies, reveal key residues involved in Mn2+ coordination, substrate binding, and catalytic reactions. We also provide a possible catalytic mechanism for this unique type of NDP-pyranose mutase. Taken together, our results highlight key elements of an enzyme family important for furanose biosynthesis.

The tRNA pseudouridine synthase TruB1 regulates the maturation of let-7 miRNA.

Let-7 is an evolutionary conserved microRNA that mediates post-transcriptional gene silencing to regulate a wide range of biological processes, including development, differentiation, and tumor suppression. Let-7 biogenesis is tightly regulated by several RNA-binding proteins, including Lin28A/B, which represses let-7 maturation. To identify new regulators of let-7, we devised a cell-based functional screen of RNA-binding proteins using a let-7 sensor luciferase reporter and identified the tRNA pseudouridine synthase, TruB1. TruB1 enhanced maturation specifically of let-7 family members. Rather than inducing pseudouridylation of the miRNAs, high-throughput sequencing crosslinking immunoprecipitation (HITS-CLIP) and biochemical analyses revealed direct binding between endogenous TruB1 and the stem-loop structure of pri-let-7, which also binds Lin28A/B. TruB1 selectively enhanced the interaction between pri-let-7 and the microprocessor DGCR8, which mediates miRNA maturation. Finally, TruB1 suppressed cell proliferation, which was mediated in part by let-7. Altogether, we reveal an unexpected function for TruB1 in promoting let-7 maturation.

Structural basis of pri-let-7 recognition by human pseudouridine synthase TruB1.

Let-7 was one of the first microRNAs (miRNAs) to be discovered and its expression promotes differentiation during development and function as tumor suppressors in various cancers. The maturation process of let-7 miRNA is tightly regulated by multiple RNA-binding proteins. For example, LIN28 binds to the terminal loops of the precursors of let-7 family and block their processing into mature miRNAs. Trim25 promotes the uridylation-mediated degradation of pre-let-7 modified by LIN28/TUT4. Recently, human pseudouridine synthase TruB1 has been reported to facilitate let-7 maturation by directly binding to pri-let-7 and recruiting Drosha-DGCR8 microprocessor. Through biochemical assay and structural investigation, we show that human TruB1 binds specifically the terminal loop of pri-let-7a1 at nucleotides 31-41, which folds as a small stem-loop architecture. Although TruB1 recognizes the terminal loop of pri-let-7a1 in a way similar to how E. coli TruB interacts with tRNA, a conserved KRKK motif in human and other higher eukaryotes adds an extra binding interface and strengthens the recognition of TruB1 for pri-let-7a1 through electrostatic interactions. These findings reveal the structural basis of TruB1-pri-let-7 interaction which may assists the elucidation of precise role of TruB1 in biogenesis of let-7.

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.

Adjusting Catalytic Activity of ß-Amyrin Synthase GgBAS by Utilizing the Plasticity Residues of an Active Site.

ß-Amyrin synthase (bAS) is a representative plant oxidosqualene cyclase (OSC), and previous studies have identified many functional residues and mutants that can alter its catalytic activity. However, the regulatory mechanism of the active site architecture for adjusting the catalytic activity remains unclear. In this study, we investigate the function of key residues and their regulatory effects on the catalytic activity of Glycyrrhiza glabra ß-amyrin synthase (GgbAS) through molecular dynamics simulations and site-directed mutagenesis experiments. We identified the plasticity residues located in two active site regions and explored the interactions between these residues and tetracyclic/pentacyclic intermediates. Based on computational and experimental results, we further categorize these plasticity residues into three types: effector, adjuster, and supporter residues, according to their functions in the catalytic process. This study provides valuable insights into the catalytic mechanism and active site plasticity of GgbAS, offering important references for the rational enzyme engineering of other OSC enzyme.

SA and NHP glucosyltransferase UGT76B1 affects plant defense in both SID2- and NPR1-dependent and independent manner.

KEY MESSAGE: The small-molecule glucosyltransferase loss-of-function mutant ugt76b1 exhibits both SID2- or NPR1-dependent and independent facets of enhanced plant immunity, whereupon FMO1 is required for the SID2 and NPR1 independence. The small-molecule glucosyltransferase UGT76B1 inactivates salicylic acid (SA), isoleucic acid (ILA), and N-hydroxypipecolic acid (NHP). ugt76b1 loss-of-function plants manifest an enhanced defense status. Thus, we were interested how UGT76B1 genetically integrates in defense pathways and whether all impacts depend on SA and NHP. We study the integration of UGT76B1 by transcriptome analyses of ugt76b1. The comparison of transcripts altered by the loss of UGT76B1 with public transcriptome data reveals both SA-responsive, ISOCHORISMATE SYNTHASE 1/SALICYLIC ACID INDUCTION DEFICIENT 2 (ICS1/SID2)- and NON EXPRESSOR OF PR GENES 1 (NPR1)-dependent, consistent with the role of UGT76B1 in glucosylating SA, and SA-non-responsive, SID2/NPR1-independent genes. We also discovered that UGT76B1 impacts on a group of genes showing non-SA-responsiveness and regulation by infections independent from SID2/NPR1. Enhanced resistance of ugt76b1 against Pseudomonas syringae is partially independent from SID2 and NPR1. In contrast, the ugt76b1-activated resistance is completely dependent on FMO1 encoding the NHP-synthesizing FLAVIN-DEPENDENT MONOOXYGENASE 1). Moreover, FMO1 ranks top among the ugt76b1-induced SID2- and NPR1-independent pathogen responsive genes, suggesting that FMO1 determines the SID2- and NPR1-independent effect of ugt76b1. Furthermore, the genetic study revealed that FMO1, ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), SID2, and NPR1 are required for the SA-JA crosstalk and senescence development of ugt76b1, indicating that EDS1 and FMO1 have a similar effect like stress-induced SA biosynthesis (SID2) or the key SA signaling regulator NPR1. Thus, UGT76B1 influences both SID2/NPR1-dependent and independent plant immunity, and the SID2/NPR1 independence is relying on FMO1 and its product NHP, another substrate of UGT76B1.

Human TRUB1 is a highly conserved pseudouridine synthase responsible for the formation of Ψ55 in mitochondrial tRNAAsn, tRNAGln, tRNAGlu and tRNAPro.

Pseudouridine (Ψ) at position 55 in tRNAs plays an important role in their structure and function. This modification is catalyzed by TruB/Pus4/Cbf5 family of pseudouridine synthases in bacteria and yeast. However, the mechanism of TRUB family underlying the formation of Ψ55 in the mammalian tRNAs is largely unknown. In this report, the CMC/reverse transcription assays demonstrated the presence of Ψ55 in the human mitochondrial tRNAAsn, tRNAGln, tRNAGlu, tRNAPro, tRNAMet, tRNALeu(UUR) and tRNASer(UCN). TRUB1 knockout (KO) cell lines generated by CRISPR/Cas9 technology exhibited the loss of Ψ55 modification in mitochondrial tRNAAsn, tRNAGln, tRNAGlu and tRNAPro but did not affect other 18 mitochondrial tRNAs. An in vitro assay revealed that recombinant TRUB1 protein can catalyze the efficient formation of Ψ55 in tRNAAsn and tRNAGln, but not in tRNAMet and tRNAArg. Notably, the overexpression of TRUB1 cDNA reversed the deficient Ψ55 modifications in these tRNAs in TRUB1KO HeLa cells. TRUB1 deficiency affected the base-pairing (18A/G-Ψ55), conformation and stability but not aminoacylation capacity of these tRNAs. Furthermore, TRUB1 deficiency impacted mitochondrial translation and biogenesis of oxidative phosphorylation system. Our findings demonstrated that human TRUB1 is a highly conserved mitochondrial pseudouridine synthase responsible for the Ψ55 modification in the mitochondrial tRNAAsn, tRNAGln, tRNAGlu and tRNAPro.

A squalene-hopene cyclase in Schizosaccharomyces japonicus represents a eukaryotic adaptation to sterol-limited anaerobic environments.

Biosynthesis of sterols, which are key constituents of canonical eukaryotic membranes, requires molecular oxygen. Anaerobic protists and deep-branching anaerobic fungi are the only eukaryotes in which a mechanism for sterol-independent growth has been elucidated. In these organisms, tetrahymanol, formed through oxygen-independent cyclization of squalene by a squalene-tetrahymanol cyclase, acts as a sterol surrogate. This study confirms an early report [C. J. E. A. Bulder, Antonie Van Leeuwenhoek, 37, 353-358 (1971)] that Schizosaccharomyces japonicus is exceptional among yeasts in growing anaerobically on synthetic media lacking sterols and unsaturated fatty acids. Mass spectrometry of lipid fractions of anaerobically grown Sch. japonicus showed the presence of hopanoids, a class of cyclic triterpenoids not previously detected in yeasts, including hop-22(29)-ene, hop-17(21)-ene, hop-21(22)-ene, and hopan-22-ol. A putative gene in Sch. japonicus showed high similarity to bacterial squalene-hopene cyclase (SHC) genes and in particular to those of Acetobacter species. No orthologs of the putative Sch. japonicus SHC were found in other yeast species. Expression of the Sch. japonicus SHC gene (Sjshc1) in Saccharomyces cerevisiae enabled hopanoid synthesis and stimulated anaerobic growth in sterol-free media, thus indicating that one or more of the hopanoids produced by SjShc1 could at least partially replace sterols. Use of hopanoids as sterol surrogates represents a previously unknown adaptation of eukaryotic cells to anaerobic growth. The fast anaerobic growth of Sch. japonicus in sterol-free media is an interesting trait for developing robust fungal cell factories for application in anaerobic industrial processes.

Tocopherol controls D1 amino acid oxidation by oxygen radicals in Photosystem II.

Photosystem II (PSII) is an intrinsic membrane protein complex that functions as a light-driven water:plastoquinone oxidoreductase in oxygenic photosynthesis. Electron transport in PSII is associated with formation of reactive oxygen species (ROS) responsible for oxidative modifications of PSII proteins. In this study, oxidative modifications of the D1 and D2 proteins by the superoxide anion (O2•-) and the hydroxyl (HO•) radicals were studied in WT and a tocopherol cyclase (vte1) mutant, which is deficient in the lipid-soluble antioxidant α-tocopherol. In the absence of this antioxidant, high-resolution tandem mass spectrometry was used to identify oxidation of D1:130E to hydroxyglutamic acid by O2•- at the PheoD1 site. Additionally, D1:246Y was modified to either tyrosine hydroperoxide or dihydroxyphenylalanine by O2•- and HO•, respectively, in the vicinity of the nonheme iron. We propose that α-tocopherol is localized near PheoD1 and the nonheme iron, with its chromanol head exposed to the lipid-water interface. This helps to prevent oxidative modification of the amino acid's hydrogen that is bonded to PheoD1 and the nonheme iron (via bicarbonate), and thus protects electron transport in PSII from ROS damage.

Probing the functions of friedelane-type triterpene cyclases from four celastrol-producing plants.

Triterpenes are among the most diverse plant natural products, and their diversity is closely related to various triterpene skeletons catalyzed by different 2,3-oxidosqualene cyclases (OSCs). Celastrol, a friedelane-type triterpene with significant bioactivities, is specifically distributed in higher plants, such as Celastraceae species. Friedelin is an important precursor for the biosynthesis of celastrol, and it is synthesized through the cyclization of 2,3-oxidosqualene, with the highest number of rearrangements being catalyzed by friedelane-type triterpene cyclases. However, the molecular mechanisms underlying the catalysis of friedelin production by friedelane-type triterpene cyclases have not yet been fully elucidated. In this study, transcriptome data of four celastrol-producing plants from Celastraceae were used to identify a total of 21 putative OSCs. Through functional characterization, the friedelane-type triterpene cyclases were separately verified in the four plants. Analysis of the selection pressure showed that purifying selection acted on these OSCs, and the friedelane-type triterpene cyclases may undergo weaker selective restriction during evolution. Molecular docking and site-directed mutagenesis revealed that changes in some amino acids that are unique to friedelane-type triterpene cyclases may lead to variations in catalytic specificity or efficiency, thereby affecting the synthesis of friedelin. Our research explored the functional diversity of triterpene synthases from a multispecies perspective. It also provides some references for further research on the relative mechanisms of friedelin biosynthesis.