Genomic and structural basis for evolution of tropane alkaloid biosynthesis.

The tropane alkaloids (TAs) cocaine and hyoscyamine have been used medicinally for thousands of years. To understand the evolutionary origins and trajectories of serial biosynthetic enzymes of TAs and especially the characteristic tropane skeletons, we generated the chromosome-level genome assemblies of cocaine-producing Erythroxylum novogranatense (Erythroxylaceae, rosids clade) and hyoscyamine-producing Anisodus acutangulus (Solanaceae, asterids clade). Comparative genomic and phylogenetic analysis suggested that the lack of spermidine synthase/N-methyltransferase (EnSPMT1) in ancestral asterids species contributed to the divergence of polyamine (spermidine or putrescine) methylation in cocaine and hyoscyamine biosynthesis. Molecular docking analysis and key site mutation experiments suggested that ecgonone synthases CYP81AN15 and CYP82M3 adopt different active-site architectures to biosynthesize the same product ecgonone from the same substrate in Erythroxylaceae and Solanaceae. Further synteny analysis showed different evolutionary origins and trajectories of CYP81AN15 and CYP82M3, particularly the emergence of CYP81AN15 through the neofunctionalization of ancient tandem duplication genes. The combination of structural biology and comparative genomic analysis revealed that ecgonone methyltransferase, which is responsible for the biosynthesis of characteristic 2-substituted carboxymethyl group in cocaine, evolved from the tandem copies of salicylic acid methyltransferase by the mutations of critical E216 and S153 residues. Overall, we provided strong evidence for the independent origins of serial TA biosynthetic enzymes on the genomic and structural level, underlying the chemotypic convergence of TAs in phylogenetically distant species.

Two Unprecedented Germanoniobate Frameworks Based on High-Nuclearity Peanut-Shaped {Ge12Nb38} Clusters.

By variation of the amount of GeO2, two organic-inorganic hybrid germanoniobate frameworks with 6-connected pcu and 10-connected bct topologies were constructed from peanut-shaped {α-Ge12Nb38} and {ß-Ge12Nb38} clusters, respectively. The {α-Ge12Nb38} and {ß-Ge12Nb38} clusters contain the most Ge centers of germanoniobates reported so far. The compounds exhibit proton conduction properties with a conductivity of 3.04 × 10-4 S·cm-3 for 1 and 1.62 × 10-4 S·cm-3 for 2 at 85 °C and 98% RH. The water vapor adsorption capacities for 1 and 2 are 5.86 and 4.40 mmol·g-1, respectively.

Identification and characterization of camptothecin tailoring enzymes in Nothapodytes tomentosa.

Camptothecin is a complex monoterpenoid indole alkaloid with remarkable antitumor activity. Given that two C-10 modified camptothecin derivatives, topotecan and irinotecan, have been approved as potent anticancer agents, there is a critical need for methods to access other aromatic ring-functionalized congeners (e.g., C-9, C-10, etc.). However, contemporary methods for chemical oxidation are generally harsh and low-yielding when applied to the camptothecin scaffold, thereby limiting the development of modified derivatives. Reported herein, we have identified four tailoring enzymes responsible for C-9 modifications of camptothecin from Nothapodytes tomentosa, via metabolomic and transcriptomic analysis. These consist of a cytochrome P450 (NtCPT9H) which catalyzes the regioselective oxidation of camptothecin to 9-hydroxycamptothecin, as well as two methyltransferases (NtOMT1/2, converting 9-hydroxycamptothecin to 9-methoxycamptothecin), and a uridine diphosphate-glycosyltransferase (NtUGT5, decorating 9-hydroxycamptothecin to 9-ß-D-glucosyloxycamptothecin). Importantly, the critical residues that contribute to the specific catalytic activity of NtCPT9H have been elucidated through molecular docking and mutagenesis experiments. This work provides a genetic basis for producing camptothecin derivatives through metabolic engineering. This will hasten the discovery of novel C-9 modified camptothecin derivatives, with profound implications for pharmaceutical manufacture.

Reversible Thermochromism and Stable Resistance Switching Behaviors Based on a Co(III)-Complex-Linked Polyoxoniobate.

A 3D Co(III)-complex hybrid polyoxoniobate framework Na10(H2O)36[Co2(phen)2(4,4'-bipy)(Nb6O19)2]·19H2O (1) has been constructed from [Co2(phen)2(4,4'-bipy)(Nb6O19)2]10- dimer units and 2D inorganic Na-O cluster layers. The Co(III) centers are coordinated with {Nb6O19}, 4,4'-bipy and phen simultaneously. The [Co2(phen)2(4,4'-bipy)(Nb6O19)2]10- fragments link the Na-O cluster layers to generate a 3D metal complex-modified hybrid polyoxoniobate framework with π-π interactions between phenanthroline rings. Compound 1 shows reversible thermochromic behavior resulting from electron transfer from {Nb6O19} to 4,4'-bipy and subsequent formation of radical products, which is first observed in polyoxoniobates. Furthermore, the compound exhibits stable nonvolatile storage behavior and rewritable resistive switching with a low switching voltage (1.12 V) and high current on/off ratio (1.18 × 103) along with stable cyclic performance during stability test for 200 cycles. Charge-transfer mechanism has been studied by analyzing the relationship between current and voltage in the process of resistance switching.

Discovery and Engineering of the Cocaine Biosynthetic Pathway.

Cocaine, the archetypal tropane alkaloid from the plant genus Erythroxylum, has recently been used clinically as a topical anesthesia of the mucous membranes. Despite this, the key biosynthetic step of the requisite tropane skeleton (methylecgonone) from the identified intermediate 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid (MPOA) has remained, until this point, unknown. Herein, we identify two missing enzymes (EnCYP81AN15 and EnMT4) necessary for the biosynthesis of the tropane skeleton in cocaine by transient expression of the candidate genes in Nicotiana benthamiana. Cytochrome P450 EnCYP81AN15 was observed to selectively mediate the oxidative cyclization of S-MPOA to yield the unstable intermediate ecgonone, which was then methylated to form optically active methylecgonone by methyltransferase EnMT4 in Erythroxylum novogranatense. The establishment of this pathway corrects the long-standing (but incorrect) biosynthetic hypothesis of MPOA methylation first and oxidative cyclization second. Notably, the de novo reconstruction of cocaine was realized in N. benthamiana with the two newly identified genes, as well as four already known ones. This study not only reports a near-complete biosynthetic pathway of cocaine and provides new insights into the metabolic networks of tropane alkaloids (cocaine and hyoscyamine) in plants but also enables the heterologous synthesis of tropane alkaloids in other (micro)organisms, entailing significant implications for pharmaceutical production.

Tropane alkaloid biosynthesis: a centennial review.

Covering: 1917 to 2020Tropane alkaloids (TAs) are a remarkable class of plant secondary metabolites, which are characterized by an 8-azabicyclo[3.2.1]octane (nortropane) ring. Members of this class, such as hyoscyamine, scopolamine, and cocaine, are well known for their long history as poisons, hallucinogens, and anaesthetic agents. Since the structure of the tropane ring system was first elucidated in 1901, organic chemists and biochemists have been interested in how these mysterious tropane alkaloids are assembled in vitro and in vivo. However, it was only in 2020 that the complete biosynthetic route of hyoscyamine and scopolamine was clarified, and their de novo production in yeast was also achieved. The aim of this review is to present the innovative ideas and results in exploring the story of tropane alkaloid biosynthesis in plants from 1917 to 2020. This review also highlights that Robinson's classic synthesis of tropinone, which is one hundred years old, is biomimetic, and underscores the importance of total synthesis in the study of natural product biosynthesis.

Luminescent 3D Lanthanide-Cadmium Heterometal-Organic Frameworks with Chemical Stability and Selective Luminescent Sensing.

Four novel three-dimensional (3D) 4d-4f heterometal-organic compounds, [LnCd2(Pbc)4(Meimdc)(H2O)]·3H2O (Ln = Eu, 1a; Tb, 1b; Sm, 1c; Dy, 1d) (HPbc = 4-(4-pyridinyl)benzoic acid; H3Meimdc = 2-methyl-1H-4,5-imidazole-dicarboxylic acid), have been successfully prepared by a hydrothermal method. All the compounds are isostructural and show three-dimensional microporous pillar-layered structures with uncoordinated carboxylate sites hung in the channels. Compound 1a possesses excellent chemical stability. The luminescent investigations show that compounds 1a, 1b, 1c, and 1d display the characteristic emission bands of Ln3+ ions. Compound 1a exhibits a good potential as a luminescent sensor material for multi-responsive Ag+, Cu2+, Zn+, Co2+, and Ni2+ cations and some organic amines. Interestingly, 1a can capture Ag+, Cu2+, Zn+, Co2+, and Ni2+ cations and shows cation-dependent colorimetric response, which indicates the potential for naked sensing.

The Anti-Allergic Rhinitis Effect of Traditional Chinese Medicine of Shenqi by Regulating Mast Cell Degranulation and Th1/Th2 Cytokine Balance.

Shenqi is a traditional Chinese polyherbal medicine has been widely used for the treatment of allergic rhinitis (AR). The aim of this study was to investigate the anti-allergic rhinitis activity of Shenqi and explore its underlying molecular mechanism. Ovalbumin (OVA)-induced allergic rhinitis rat model was used to evaluate the anti-allergic rhinitis effect of Shenqi. The effect of Shenqi on IgE-mediated degranulation was measured using rat basophilic leukemia (RBL-2H3) cells. Primary spleen lymphocytes were isolated to investigate the anti-allergic mechanism of Shenqi by detecting the expression of transcription factors via Western blot and the level of cytokines (IL-4 and IFN-γ) via ELISA. In OVA-induced AR rat models, Shenqi relieved the allergic rhinitis symptoms, inhibited the histopathological changes of nasal mucosa, and reduced the levels of IL-4 and IgE. The results from the in vitro study certified that Shenqi inhibited mast cell degranulation. Furthermore, the results of GATA3, T-bet, p-STAT6, and SOCS1 expression and production of IFN-γ and IL-4 demonstrated that Shenqi balanced the ratio of Th1/Th2 (IFN-γ/IL-4) in OVA-stimulated spleen lymphocytes. In conclusion, these results suggest that Shenqi exhibits an obvious anti-allergic effect by suppressing the mast cell-mediated allergic response and by improving the imbalance of Th1/Th2 ratio in allergic rhinitis.

Chromosome level genome assembly of endangered medicinal plant Anisodus tanguticus.

Anisodus tanguticus is a medicinal herb that belongs to the Anisodus genus of the Solanaceae family. This endangered herb is mainly distributed in Qinghai-Tibet Plateau. In this study, we combined the Illumina short-read, Nanopore long-read and high-throughput chromosome conformation capture (Hi-C) sequencing technologies to de novo assemble the A. tanguticus genome. A high-quality chromosomal-level genome assembly was obtained with a genome size of 1.26 Gb and a contig N50 of 25.07 Mb. Of the draft genome sequences, 97.47% were anchored to 24 pseudochromosomes with a scaffold N50 of 51.28 Mb. In addition, 842.14 Mb of transposable elements occupying 66.70% of the genome assembly were identified and 44,252 protein-coding genes were predicted. The genome assembly of A. tanguticus will provide genetic repertoire to understand the adaptation strategy of Anisodus species in the plateau, which will further promote the conservation of endangered A. tanguticus resources.

Guvermectin Biosynthesis Revealing the Key Role of a Phosphoribohydrolase and Structural Insight into the Active Glutamate of a Noncanonical Adenine Phosphoribosyltransferase.

Guvermectin is a novel plant growth regulator that has been registered as a new agrochemical in China. It is an adenosine analogue with an unusual psicofuranose instead of ribose. Herein, the gene cluster responsible for guvermectin biosynthesis in Streptomyces caniferus NEAU6 is identified using gene interruption and heterologous expression experiments. A key intermediate psicofuranine 6'-phosphate (PMP) is chemically synthesized, and the functions of GvmB, C, D, and E are verified by individual stepwise enzyme reactions in vitro. The results also show that the biosynthesis of guvermectin is coupled with adenosine production by a single cluster. The higher catalytic efficiency of GvmB on PMP than AMP ensures the effective biosynthesis of guvermectin. Moreover, a phosphoribohydrolase GvmA is employed in the pathway that can hydrolyze AMP but not PMP and shows higher catalytic efficiency for the AMP hydrolysis than that of the AMP dephosphorylation by GvmB, leading to shunting of adenosine biosynthesis toward the production of guvermectin. Finally, the crystal structure of GvmE in complex with the product PMP has been solved. Glu160 at the C-terminal is identified as the acid/base for protonation/deprotonation of N7 of the adenine ring, demonstrating that GvmE is a noncanonical adenine phosphoribosyltransferase.

Two high-nuclearity isopolyoxoniobates containing {Nb54 O151}-based helical nanotubes for the decomposition of chemical warfare agent simulants.

Two inorganic-organic hybrid isopolyoxoniobates (1 and 2) based on new high-nuclearity {Nb54O151} clusters have been synthesized under hydrothermal conditions. In particular, the combination of the unique {Nb54O151} clusters with copper-amine complexes has led to rare helical nanotubes, which are further linked by alkali metal cations or copper-amine complexes into two 2D similar bamboo-raft-like layered networks (1 and 2), respectively. Compound 1 exhibits effective base-catalytic decomposition of chemical warfare agent simulants dimethyl methylphosphonate (DMMP) and diethyl cyanophosphonate (DECP).

Advances in chemistry and bioactivity of the genus Erythroxylum.

Erythroxylum P. Browne is the largest and most representative genus of Erythroxylaceae family. It contains approximately 230 species that are mainly distributed in tropical and subtropical regions. Some species in this genus, such as E. monogynum and E. coca, have been used as folk medicines in India or South America for a long history. It is well known that Erythroxylum plants are rich in tropane alkaloids, and the representative member cocaine shows remarkable activity in human central nervous system. However, many other types of active compounds have also been found in Erythroxylum along with the broadening and deepening of phytochemical research. To date, a total of 383 compounds from Erythroxylum have been reported, among which only 186 tropane alkaloids have been reviewed in 2010. In this review, we summarized all remained 197 compounds characterized from 53 Erythroxylum species from 1960 to 2021, which include diterpenes, triterpenes, alkaloids, flavonoids, and other derivates, providing a comprehensive overview of phytoconstituents profile of Erythroxylum plants. In addition, the biological activities of representative phytochemicals and crude extracts were also highlighted.

Catalytic innovation underlies independent recruitment of polyketide synthases in cocaine and hyoscyamine biosynthesis.

Tropane alkaloids such as hyoscyamine and cocaine are of importance in medicinal uses. Only recently has the hyoscyamine biosynthetic machinery become complete. However, the cocaine biosynthesis pathway remains only partially elucidated. Here we characterize polyketide synthases required for generating 3-oxo-glutaric acid from malonyl-CoA in cocaine biosynthetic route. Structural analysis shows that these two polyketide synthases adopt distinctly different active site architecture to catalyze the same reaction as pyrrolidine ketide synthase in hyoscyamine biosynthesis, revealing an unusual parallel/convergent evolution of biochemical function in homologous enzymes. Further phylogenetic analysis suggests lineage-specific acquisition of polyketide synthases required for tropane alkaloid biosynthesis in Erythroxylaceae and Solanaceae species, respectively. Overall, our work elucidates not only a key unknown step in cocaine biosynthesis pathway but also, more importantly, structural and biochemical basis for independent recruitment of polyketide synthases in tropane alkaloid biosynthesis, thus broadening the understanding of conservation and innovation of biosynthetic catalysts.

Characterization of inthomycin biosynthetic gene cluster revealing new insights into carboxamide formation.

Inthomycins are polyketide antibiotics which contain a terminal carboxamide group and a triene chain. Inthomycin B (1) and its two new analogues 2 and 3 were isolated from the crude extract of Streptomyces pactum L8. Identification of the gene cluster for inthomycin biosynthesis as well as the 15N-labeled glycine incorporation into inthomycins are described. Combined with the gene deletion of the rare P450 domain in the NRPS module, a formation mechanism of carboxamide moiety in inthomycins was proposed via an oxidative release of the assembly chain assisted by the P450 domain.

Cangumycins A-F, six new angucyclinone analogues with immunosuppressive activity from Streptomyces.

Cangumycins A-F (1-6), six new angucyclinone analogues, together with two known ones (7 and 8), were isolated from the fermentation broth of a soil-derived Streptomyces sp. KIB-M10. Structures of these compounds were elucidated via a joint use of spectroscopic analyses and single-crystal X-ray diffractions. Among them, cangumycins E (5) and F (6) share a C-ring cleaved backbone, and cangumycins B (2) and E (5) exhibit potent immunosuppressive activity (IC50 8.1 and 2.7 µmol·L-1, respectively) against human T cell proliferation at a non-cytotoxic concentration.

Octahedron-shaped three-shell Ln14-substituted polyoxotungstogermanates encapsulating a W4O15 cluster: luminescence and frequency dependent magnetic properties.

A series of new Ln14-substituted polyoxometalates, H27Na16[(Ln14(H2O)W4(OH)O14)(WO4)4(GeW10O38)6]·nH2O (n ≈ 86) (1-Ln, Ln = Eu, Gd, Tb, Dy, Ho, Er), which contain 14 Ln3+ and 6 {α(1,5)-[GeW10O38]12-} units, has been synthesized in one-pot reactions. As the first 14-Ln-containing polyoxometalates, the octahedron-shaped three-shell structure of the [(Ln14(H2O)W4(OH)O14)(WO4)4(GeW10O38)6]43- polyanion is made up of a W4 tetrahedron, a Ln14W4 tetrahedron and a Ge6W60 octahedron. Compound 1-Dy exhibits frequency dependent magnetic properties. Compounds 1-Eu, 1-Tb and 1-Dy exhibit the characteristic emission bands of the Ln3+ ion.

NIR emission and luminescent sensing of a lanthanide-organic framework with Lewis basic imidazole and pyridyl sites.

A series of lanthanide-organic frameworks [Ln(Himdc)(ina)(H2O)]n (Ln = Eu 1a; Sm 1b; and Nd 1c, H3imdc = imidazole-4,5-dicarboxylic acid, Hina = isonicotinic acid) were synthesized under hydrothermal conditions. All compounds 1a-1c consist of a 3D microporous lanthanide carboxylate ([Eu(COO)3]n) framework and uncoordinated Lewis basic pyridyl and imidazole groups hung in the channels. The luminescence investigations show that the compound 1c displays an interesting NIR luminescence property. The compound 1a exhibits a good potential as a luminescent multi-responsive sensing material for Fe3+ ions and Cr2O72- anions.

[Analysis of anthropometry on head and eye for stipulating of children's spectacle frames].

OBJECTIVE: To investigate the normal value of six parameters of head and eye in Chinese children, including interorbit distance (IOD), interpupillary distance (IPD), superior otobasion-ectocochion distance (obs-ec), intersuperior otobasion distance (obs-obs), exophthalmos and nasal basis angle, for stipulating the sizes of children spectacle frames. METHODS: The value of these six parameters in 10 171 children ages 5 to 17 years old were measured with caliper in four cities and the results were analyzed. RESULTS: The IOD, IPD, obs-ec and obs-obs were increased and positively correlated with the age. The differences of these parameters among different ages were statistically significant (F = 0.97, P < 0.01), especially in IPD before 15, IOD before 13, obs-ec and obs-obs before 12 and exophthalmos before 12 years old. The fastest growth age in IPD, IOD, exophthalmos, obs-ec and obs-obs was 10, 11, 5, 7 and 11 years old, respectively. The growth period of IPD, IOD, exophthalmos, obs-ec and obs-obs was 5 - 15, 5 - 13, 5 - 8, 5 - 12 and 5 - 15 years old. The difference of these six parameters among children in 4 cities were statistically significant (t = 3.1, P < 0.01). CONCLUSIONS: The most active growth period of IPD, IOD, obs-obs, obs-ec and exophthalmos lies between 5-15 years old in Chinese children 5-17 years old. Although the differences of these six parameters between male and female and among children in four cities are statistically significant, practically they are not important for stipulating the sizes of children's spectacle frames.

[Study on chemical constituents in total saponin from Trigonella foenum-graecum].

OBJECTIVE: To study the chemical constituents in the total saponin from Trigonellf foenum-graecum. METHOD: The compounds were isolated by column chromatography on macroporous resin and silica gel and elucidated by physical and chemical evidences and spectroscopic analysis. RESULT: Two compounds were obtained and identifiedas methyl-protodioscin and methyl-protodeltonin. CONCLUSION: Methyl-protodioscin and methyl-protodeltonin were isolated from this plant for the first time.

Effect of quaternization degree on physiochemical and biological activities of chitosan from squid pens.

Chitosan was prepared by alkaline N-deacetylation of ß-chitin from squid pens, and N-(2-hydroxy) propyl-3-trimethyl ammonium chitosan chloride (HTCC) derivatives, with different degrees of quaternization (DQ) ranging from 0.77 to 1.06, were synthesized. It was identified by FT-IR, 1H NMR and XRD analysis. All of the HTCC showed good water solubility in a wide pH range. The moisture absorption and retention abilities of all the HTCC were much better than that of the chitosan. The moisture absorption and retention values of all the HTCC at 43% RH for 24 h were above 49% and 92%, respectively. The scavenging ability of HTCC against hydroxyl and ABTS radicals improved with increasing concentration. The effectiveness of HTCC against hydroxyl radicals was lower than that of chitosan. These results indicated that HTCC, which has a much better moisture absorption and retention capacity, may act as a potential moisturizer in vitro.