Molecular cloning and functional characterization of 2,3-oxidosqualene cyclases from Artemisia argyi.

Artemisia argyi is a traditional medicinal and edible plant, generating various triterpenoids with pharmacological activities, such as anti-virus, anti-cancer, and anti-oxidant. The 2,3-oxidosqualene cyclase family of A. argyi offers novel insights into the triterpenoid pathway, which might contribute to the medicinal value of its tissue extracts. Nevertheless, the biosynthesis of active triterpenoids in Artemisia argyi is still uncertain. In this study, four putative OSC (2,3-oxidosqualene cyclase) genes (AaOSC1-4) were first isolated and identified from A. argyi. Through the yeast heterologous expression system, three AaOSCs were characterized for the biosynthesis of diverse triterpenoids including cycloartenol, ß-amyrin, (3S,13R)-malabarica-14(27),17,21-trien-3ß-ol, and dammara-20,24-dien-3ß-ol. AaOSC1 was a multifunctional dammara-20,24-dien-3ß-ol synthase, which yielded 8 different triterpenoids, including tricyclic, and tetracyclic products. AaOSC2 and AaOSC3 were cycloartenol, and ß-amyrin synthases, respectively. As a result, these findings provide a deeper understanding of the biosynthesis pathway of triterpenes in A. argyi.

Biocatalytic stereocontrolled head-to-tail cyclizations of unbiased terpenes as a tool in chemoenzymatic synthesis.

Terpene synthesis stands at the forefront of modern synthetic chemistry and represents the state-of-the-art in the chemist's toolbox. Notwithstanding, these endeavors are inherently tied to the current availability of natural cyclic building blocks. Addressing this limitation, the stereocontrolled cyclization of abundant unbiased linear terpenes emerges as a valuable tool, which is still difficult to achieve with chemical catalysts. In this study, we showcase the remarkable capabilities of squalene-hopene cyclases (SHCs) in the chemoenzymatic synthesis of head-to-tail-fused terpenes. By combining engineered SHCs and a practical reaction setup, we generate ten chiral scaffolds with >99% ee and de, at up to decagram scale. Our mechanistic insights suggest how cyclodextrin encapsulation of terpenes may influence the performance of the membrane-bound enzyme. Moreover, we transform the chiral templates to valuable (mero)-terpenes using interdisciplinary synthetic methods, including a catalytic ring-contraction of enol-ethers facilitated by cooperative iodine/lipase catalysis.

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.

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.

Genome-Wide Investigation of Oxidosqualene Cyclase Genes Deciphers the Genetic Basis of Triterpene Biosynthesis in Tea Plants.

Triterpenoids from Camellia species comprise a diverse class of bioactive compounds with great therapeutic potential. However, triterpene biosynthesis in tea plants (Camellia sinensis) remains elusive. Here, we identified eight putative 2,3-oxidosqualene cyclase (OSC) genes (CsOSC1-8) from the tea genome and characterized the functions of five through heterologous expression in yeast and tobacco and transient overexpression in tea plants. CsOSC1 was found to be a ß-amyrin synthase, whereas CsOSC4, 5, and 6 exhibited multifunctional α-amyrin synthase activity. Molecular docking and site-directed mutagenesis showed that the CsOSC6M259T/W260L double mutant yielded >40% lupeol, while the CsOSC1 W259L single mutant alone was sufficient for lupeol production. The V732F mutation in CsOSC5 altered product formation from friedelin to taraxasterol and ψ-taraxasterol. The L254 M mutation in the cycloartenol synthase CsOSC8 enhanced the catalytic activity. Our findings shed light on the molecular basis governing triterpene diversity in tea plants and offer potential avenues for OSC engineering.

Bacteria at Work - Experimental and Theoretical Studies Reveal the Catalytic Mechanism of Ectoine Synthase.

Ectoine synthase (EctC) catalyses the ultimate step of ectoine biosynthesis, a kosmotropic compound produced as compatible solute by many bacteria and some archaea or eukaryotes. EctC is an Fe2+-dependent homodimeric cytoplasmic protein. Using Mössbauer spectroscopy, molecular dynamics simulations and QM/MM calculations, we determined the most likely coordination number and geometry of the Fe2+ ion and proposed a mechanism of the EctC-catalysed reaction. Most notably, we show that apart from the three amino acids binding to the iron ion (Glu57, Tyr84 and His92), one water molecule and one hydroxide ion are required as additional ligands for the reaction to occur. They fill the first coordination sphere of the Fe2+-cofactor and act as critical proton donors and acceptors during the cyclization reaction.

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.

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.

Evidence for the Chemical Mechanism of RibB (3,4-Dihydroxy-2-butanone 4-phosphate Synthase) of Riboflavin Biosynthesis.

RibB (3,4-dihydroxy-2-butanone 4-phosphate synthase) is a magnesium-dependent enzyme that excises the C4 of d-ribulose-5-phosphate (d-Ru5P) as formate. RibB generates the four-carbon substrate for lumazine synthase that is incorporated into the xylene moiety of lumazine and ultimately the riboflavin isoalloxazine. The reaction was first identified by Bacher and co-workers in the 1990s, and their chemical mechanism hypothesis became canonical despite minimal direct evidence. X-ray crystal structures of RibB typically show two metal ions when solved in the presence of non-native metals and/or liganding non-substrate analogues, and the consensus hypothetical mechanism has incorporated this cofactor set. We have used a variety of biochemical approaches to further characterize the chemistry catalyzed by RibB from Vibrio cholera (VcRibB). We show that full activity is achieved at metal ion concentrations equal to the enzyme concentration. This was confirmed by electron paramagnetic resonance of the enzyme reconstituted with manganese and crystal structures liganded with Mn2+ and a variety of sugar phosphates. Two transient species prior to the formation of products were identified using acid quench of single turnover reactions in combination with NMR for singly and fully 13C-labeled d-Ru5P. These data indicate that dehydration of C1 forms the first transient species, which undergoes rearrangement by a 1,2 migration, fusing C5 to C3 and generating a hydrated C4 that is poised for elimination as formate. Structures determined from time-dependent Mn2+ soaks of VcRibB-d-Ru5P crystals show accumulation in crystallo of the same intermediates. Collectively, these data reveal for the first time crucial transient chemical states in the mechanism of RibB.

Exploring the mechanism of action of lysine 5,6-aminomutase using EPR and ENDOR spectroscopies.

Radical enzymes orchestrate challenging chemical transformations by devising strategies to tame the highly reactive radical intermediates. Electron paramagnetic resonance (EPR) spectroscopy is the most suitable technique to study various aspects of the radical enzymes. Lysine 5,6-aminomutase (5,6-LAM) is one such radical enzyme and employs coenzyme B12 and pyridoxal 5'-phosphate (PLP) to catalyze the 1,2-amino shift reaction through a radical mechanism. 5,6-LAM accepts either d-lysine or l-ß-lysine as the substrate. EPR and electron nuclear double resonance (ENDOR) spectroscopies have played major roles in deciphering the mechanism of action of 5,6-LAM, while density functional theoretical (DFT) computation and synthetic isotopologues have played supporting roles. This comprehensive toolkit has revealed that 5,6-LAM undergoes large-scale conformational movement to bring PLP and coenzyme B12 close together, which allows the reaction to progress. The conformational change also closes the active site, which protects the radical intermediates and enables their transformation to product without unwanted side reactions. The substrate-related radical (S•), which is spin-coupled with Co2+ generated from homolysis of the CoC bond in coenzyme B12, was unequivocally characterized when a substrate analog, 4-thia-l-lysine, and isotopologues of it were reacted with 5,6-LAM. Studies with substrate analogs revealed a unique "odd-even" correlation with opening of the closed state. Moreover, mutagenesis studies identified the contributions that conserved residues in 5,6-LAM make toward binding of the substrate. Further studies with a cofactor analog, PLP-N-oxide, have shed light on various aspects of the mechanism of action of 5,6-LAM.

The Nitrogen Atom of Vitamin B6 Is Essential for the Catalysis of Radical Aminomutases.

Radical aminomutases are pyridoxal 5'-phosphate (PLP, a B6 vitamer)-dependent enzymes that require the generation of a 5'-deoxyadenosyl radical to initiate the catalytic cycle, to perform a 1,2 amino group shift reaction. The role of the nitrogen atom of PLP in radical aminomutases has not been investigated extensively yet. We report an alternative synthetic procedure to provide easy access to 1-deazaPLP (dAPLP), an isosteric analog of PLP which acts as a probe for studying the role of the nitrogen atom. Our results revealed that lysine 5,6-aminomutase (5,6-LAM), a radical aminomutase, reconstituted with dAPLP cannot turn over a substrate, demonstrating that the nitrogen atom is essential for radical aminomutases. In contrast, biochemical and spectroscopic studies on the S238A variant reconstituted with PLP revealed a minuscule loss of activity. This apparent anomaly can be explained by a water-mediated rescue of activity in S238A, as if mimicking the active site of lysine 2,3-aminomutase. This study leads to a better comprehension of how enzymes harness the optimum capability of PLP to realize catalysis.

[Oxidosqualene cyclases in triterpenoids biosynthesis: a review].

Triterpenoids are one of the most diverse compounds in plant metabolites, and they have a wide variety of physiological activities and are of important economic value. Oxidosqualene cyclases catalyze the cyclization of 2, 3-oxidosqualene to generate different types of sterols and plant triterpenoids, which is of great significance to the structural diversity of natural products. However, the mechanism of the diversified cyclization of 2, 3-oxidosqualene catalyzed by oxidosqualene cyclases remains unclear. This review summarized the research progress of oxidosqualene cyclases from the aspects of catalytic function, molecular evolutionary relationship between genes and proteins, protein structure, molecular simulation and molecular calculations, which may provide a reference for protein engineering and metabolic engineering of triterpene cyclase.

Computational identification of Plasmodium falciparum RNA pseudouridylate synthase as a viable drug target, its physicochemical properties, 3D structure prediction and prediction of potential inhibitors.

The increased resistance to the currently effective antimalarial drugs against Plasmodium falciparum has necessitated the development of new drugs for malaria treatment. Many proteins have been predicted using various means as potential drug targets for the treatment of the P. falciparum malaria infection. Meanwhile, only a few studies went on to predict the 3-dimensional (3D) structure of potential target. Therefore, this study aimed to predict potential antimalarial drug targets against the deadliest malaria parasite P. falciparum as well as to determine the 3D structure and possible inhibitors of one of the targets. We employed machine learning approach to predict suitable drug targets in P. falciparum. Five of the predicted protein targets were considered as potential drug targets as they were non-homologous to their human counterparts. Out of these, we determined the physicochemical properties, predicted the 3D structure and carried out docking-based virtual screening of P. falciparum RNA pseudouridylate synthase, putative (PfRPuSP). The PfRPuSP was one of the potential five target proteins. Homology modelling and the ab initio methods were used to predict the 3D structure of PfRPuSP. Then, a compound library of 5621 molecules was constructed from PubChem and ChEMBL databases using 5-fluorouridine as the control inhibitor. Docking-based virtual screening was performed using Autodock 4.2 and Autodock Vina to select compounds with high binding affinity. A total of 11 compounds were selected based on their binding energies from 881 compounds which were manually examined after docking. Seven of the 11 compounds that exhibited remarkable interactions with the residues in the active sites of PfRPuSP were analysed. These compounds performed favourably when compared to the control inhibitor and predicted to bind better than 5-fluorouridine. These seven compounds are suggested as new potential lead structures for antimalarial treatment.

The human pseudouridine synthase PUS7 recognizes RNA with an extended multi-domain binding surface.

The human pseudouridine synthase PUS7 is a versatile RNA modification enzyme targeting many RNAs thereby playing a critical role in development and brain function. Whereas all target RNAs of PUS7 share a consensus sequence, additional recognition elements are likely required, and the structural basis for RNA binding by PUS7 is unknown. Here, we characterize the structure-function relationship of human PUS7 reporting its X-ray crystal structure at 2.26 Å resolution. Compared to its bacterial homolog, human PUS7 possesses two additional subdomains, and structural modeling studies suggest that these subdomains contribute to tRNA recognition through increased interactions along the tRNA substrate. Consistent with our modeling, we find that all structural elements of tRNA are required for productive interaction with PUS7 as the consensus sequence of target RNA alone is not sufficient for pseudouridylation by human PUS7. Moreover, PUS7 binds several, non-modifiable RNAs with medium affinity which likely enables PUS7 to screen for productive RNA substrates. Following tRNA modification, the product tRNA has a significantly lower affinity for PUS7 facilitating its dissociation. Taken together our studies suggest a combination of structure-specific and sequence-specific RNA recognition by PUS7 and provide mechanistic insight into its function.

Site-directed mutagenesis and substrate compatibility to reveal the structure-function relationships of plant oxidosqualene cyclases.

Covering: up to May 2020Oxidosqualene cyclases (OSCs) catalyze one of the most complex polycyclization reactions in nature, using the linear 2,3-oxidosqualene to generate an array of triterpene skeletons in plants. Despite the structural diversity of the products, the protein sequences of plant OSCs are highly conserved, where a few key amino acids could govern the product selectivity. Due to the absence of crystal structures, site-directed mutagenesis and substrate structural modification become key approaches to understand the cyclization mechanism. In this review, 98 mutation sites in 25 plant OSCs have been summarized, and the conserved key residues have been identified by sequence alignment. Structure-function relationships are further discussed. Meanwhile, the substrate selectivity has been summarized to probe the active site cavity of plant OSCs. A total of 77 references are included.

Identification of inhibitors of UDP-galactopyranose mutase via combinatorial in situ screening.

An in situ screening assay for UDP-galactopyranose mutase (UGM, an essential enzyme of M. tuberculosis cell wall biosynthesis) has been developed to discover novel UGM inhibitors. The approach is based on the amide-forming reaction of an amino acid core with various cinnamic acids, followed by a direct fluorescence polarization assay to identify the best UGM binders without isolation and purification of the screened ligands. This assay allows us to perform one-pot high-throughput synthesis and screening of enzyme inhibitors in a 384-well plate format. UGM ligands were successfully identified by this technology and their inhibition levels were established from pure synthetic compounds in vitro and in a whole cell antibacterial assay. This study provides a blueprint for designing enamide structures as new UGM inhibitors and anti-mycobacterial agents.

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.

Targeting PUS7 suppresses tRNA pseudouridylation and glioblastoma tumorigenesis.

Pseudouridine is the most frequent epitranscriptomic modification. However, its cellular functions remain largely unknown. Here, we show that pseudouridine synthase 7 (PUS7) is highly expressed in glioblastoma versus normal brain tissues, and high PUS7 expression levels are associated with worse survival in patients with glioblastoma. PUS7 expression and catalytic activity are required for glioblastoma stem cell (GSC) tumorigenesis. Mechanistically, we identify PUS7 targets in GSCs through small RNA pseudouridine sequencing and show that pseudouridylation of PUS7-regulated transfer RNA is critical for codon-specific translational control of key regulators of GSCs. Moreover, we identify chemical inhibitors for PUS7 and show that these compounds prevent PUS7-mediated pseudouridine modification, suppress tumorigenesis and extend the life span of tumor-bearing mice. Overall, we identify an epitranscriptomic regulatory mechanism in glioblastoma and provide preclinical evidence of a potential therapeutic strategy for glioblastoma.

An Unprecedented Ring-Contraction Mechanism in Cobalamin-Dependent Radical S-Adenosylmethionine Enzymes.

A unique member of the family of cobalamin (Cbl)-dependent radical S-adenosylmethionine (SAM) enzymes, OxsB, catalyzes the ring constriction of deoxyadenosine triphosphate (dATP) to the base oxetane aldehyde phosphate, a crucial precursor for oxetanocin A (OXT-A), which is an antitumor, antiviral, and antibacterial compound. This enzyme reveals a new catalytic function for this big family that is different from the common methylation. On the basis of density functional theory calculations, a mechanism has been proposed to mainly include that the generation of 5'-deoxyadenosine radical, a hydrogen transfer forming 2'-dATP radical, and a Cbl-catalyzed ring contraction of the deoxyribose in 2'-dATP radical. The ring contraction is a concerted rearrangement step accompanied by an electron transfer from the deoxyribose hydroxyl oxygen to CoIII without any ring-opening intermediate. CoIICbl has been ruled out as an active state. Other mechanistic characteristics are also revealed. This unprecedented non-methylation mechanism provides a new catalytic repertoire for the family of radical SAM enzymes, representing a new class of ring-contraction enzymes.

In Vitro Enzymatic Conversion of Glibenclamide Using Squalene Hopene Cyclase from Pseudomonas mendocina Expressed in E. coli BL21 (DE3).

Squalene hopene cyclases catalyse the conversion of a linear substrate squalene to a cyclic product with high stereo-selectivity.The enzyme squalene hopene cyclase from Pseudomonas mendocina expressed in E. coli BL21 (DE3) was evaluated for its synthetic drug transforming ability. Nine synthetic drugs were selected as substrates for biotransformation reactions by the enzyme. The homology modelling of the protein and docking of the selected ligands were performed using GOLD suite docking software. The drug which showed maximum binding with the active-site residues of the enzyme was selected for biotransformation studies. On transformation with the enzyme, Glibenclamide, the selected antidiabetic drug alone showed significant changes in the FT/IR spectra; hence, it was selected for LCMS analysis to confirm the transformations. From the chromatogram and MS spectra, the mono-oxygenation of the product due to the enzymatic activity was confirmed. The drug transforming ability of the purified SHC could be used as an ideal tool for the generation of new and active substrate derivatives.