Plant carbonic anhydrase-like enzymes in neuroactive alkaloid biosynthesis.

Plants synthesize numerous alkaloids that mimic animal neurotransmitters1. The diversity of alkaloid structures is achieved through the generation and tailoring of unique carbon scaffolds2,3, yet many neuroactive alkaloids belong to a scaffold class for which no biosynthetic route or enzyme catalyst is known. By studying highly coordinated, tissue-specific gene expression in plants that produce neuroactive Lycopodium alkaloids4, we identified an unexpected enzyme class for alkaloid biosynthesis: neofunctionalized α-carbonic anhydrases (CAHs). We show that three CAH-like (CAL) proteins are required in the biosynthetic route to a key precursor of the Lycopodium alkaloids by catalysing a stereospecific Mannich-like condensation and subsequent bicyclic scaffold generation. Also, we describe a series of scaffold tailoring steps that generate the optimized acetylcholinesterase inhibition activity of huperzine A5. Our findings suggest a broader involvement of CAH-like enzymes in specialized metabolism and demonstrate how successive scaffold tailoring can drive potency against a neurological protein target.

The Therapeutic Effects of Seven Lycopodium Compounds on Cell Models of Alzheimer's Disease.

BACKGROUND: As an acetylcholinesterase inhibitor (AChEI), Huperzine-A (Hup-A) is marketed for treatment of mild to moderate Alzheimer's disease (AD) for decades in China. However, Hup-A causes some side effects. To search for new analogs or derivatives of Hup-A, we produced five Lycopodium alkaloids and two analogues by chemical synthesis: Lyconadins A-E, H-R-NOB, and 2JY-OBZ4. OBJECTIVE: To systematically evaluate the therapeutic effects of the seven compounds on AD cell models. METHODS: We assessed the effects of the seven compounds on cell viability via CCK-8 kit and used HEK293-hTau cells and N2a-hAPP cells as AD cell models to evaluate their potential therapeutic effects. We examined their effects on cholinesterase activity by employing the mice primary neuron. RESULTS: All compounds did not affect cell viability; in addition, Lyconadin A and 2JY-OBZ4 particularly increased cell viability. Lyconadin D and Lyconadin E restored tau phosphorylation at Thr231, and H-R-NOB and 2JY-OBZ4 restored tau phosphorylation at Thr231 and Ser396 in GSK-3ß-transfected HEK293-hTau cells. 2JY-OBZ4 decreased the level of PP2Ac-pY307 and increased the level of PP2Ac-mL309, supporting that 2JY-OBZ4 may activate PP2A. Lyconadin B, Lyconadin D, Lyconadin E, H-R-NOB, and 2JY-OBZ4 increased sAßPPα level in N2a-hAPP cells. 2JY-OBZ4 decreased the levels of BACE1 and sAßPPß, thereby reduced Aß production. Seven compounds exhibited weaker AChE activity inhibition efficiency than Hup-A. Among them, 2JY-OBZ4 showed the strongest AChE inhibition activity with an inhibition rate of 17% at 10µM. CONCLUSION: Among the seven Lycopodium compounds, 2JY-OBZ4 showed the most expected effects on promoting cell viability, downregulating tau hyperphosphorylation, and Aß production and inhibiting AChE in AD.

A metabolic regulon reveals early and late acting enzymes in neuroactive Lycopodium alkaloid biosynthesis.

Plants synthesize many diverse small molecules that affect function of the mammalian central nervous system, making them crucial sources of therapeutics for neurological disorders. A notable portion of neuroactive phytochemicals are lysine-derived alkaloids, but the mechanisms by which plants produce these compounds have remained largely unexplored. To better understand how plants synthesize these metabolites, we focused on biosynthesis of the Lycopodium alkaloids that are produced by club mosses, a clade of plants used traditionally as herbal medicines. Hundreds of Lycopodium alkaloids have been described, including huperzine A (HupA), an acetylcholine esterase inhibitor that has generated interest as a treatment for the symptoms of Alzheimer's disease. Through combined metabolomic profiling and transcriptomics, we have identified a developmentally controlled set of biosynthetic genes, or potential regulon, for the Lycopodium alkaloids. The discovery of this putative regulon facilitated the biosynthetic reconstitution and functional characterization of six enzymes that act in the initiation and conclusion of HupA biosynthesis. This includes a type III polyketide synthase that catalyzes a crucial imine-polyketide condensation, as well as three Fe(II)/2-oxoglutarate-dependent dioxygenase (2OGD) enzymes that catalyze transformations (pyridone ring-forming desaturation, piperidine ring cleavage, and redox-neutral isomerization) within downstream HupA biosynthesis. Our results expand the diversity of known chemical transformations catalyzed by 2OGDs and provide mechanistic insight into the function of noncanonical type III PKS enzymes that generate plant alkaloid scaffolds. These data offer insight into the chemical logic of Lys-derived alkaloid biosynthesis and demonstrate the tightly coordinated coexpression of secondary metabolic genes for the biosynthesis of medicinal alkaloids.

Deciphering the Biosynthetic Mechanism of Pelletierine in Lycopodium Alkaloid Biosynthesis.

Pelletierine, a proposed building block of Lycopodium alkaloids (LAs), was demonstrated to be synthesized via the non-enzymatic Mannich-like condensation of Δ1-piperideine and 3-oxoglutaric acid produced by two new type III PKSs (HsPKS4 and PcPKS1) characterized from Huperzia serrata and Phlegmariurus cryptomerianus, respectively. The findings provide new insights for further understanding the biosynthesis of LAs such as huperzine A.

Analysis of Cell Division Frequency in the Root Apical Meristem of Lycophytes, Non-seed Vascular Plants, Using EdU Labeling.

The organization of the root apical meristem (RAM) provides insights into the evolution of roots in vascular plants. The RAM of seed plants has a quiescent center (QC), in which the cells divide infrequently and function to maintain neighboring stem cells. However, the existence of a QC and the mechanisms of RAM maintenance in non-seed plants are poorly understood. We analyzed the RAM organization of lycophytes focusing on cell division activity using the EdU labeling method and showed that the RAM of Lycopodium species has a region with a very low cell division frequency, which was named the QC-like region. Here, we describe an in situ EdU labeling method for the RAM of growing roots in nature.

Inhibitory effects of serratene-type triterpenoids from Lycopodium complanatum on cholinesterases and ß-secretase 1.

Phytochemical investigation of Lycopodium complanatum whole plants led to the isolation of two new serratene-type triterpenoids (1 and 2) along with eight known triterpenoids (3-10). Their structures were established using 1D and 2D NMR spectroscopic techniques and mass spectrometry. These compounds did not inhibit acetylcholinesterases (AChE) and butyrylcholinesterase (BChE), but did inhibit ß-secretase 1 (BACE1). Compounds 1 and 6 showed potent BACE1 inhibition with IC50 values of 2.79 ± 0.28 and 2.49 ± 0.12 µM, respectively. The kinetic study of BACE1 inhibition revealed that compound 1 showed competitive inhibition, whereas 6 showed mixed-type inhibition. Furthermore, molecular docking results showed that the tested inhibitors 1 and 6 exhibited good binding affinities toward BACE1, with binding energies of -8.8 and -10.3 kcal/mol, respectively.

Lysine-derived Alkaloids: Overview and Update on Biosynthesis and Medicinal Applications with Emphasis on Quinolizidine Alkaloids.

BACKGROUND: Plants produce a vast variety of specialized metabolites which can be a rich source for lead compounds for the development of new drugs. Alkaloids are one the largest groups of plant specialized metabolites important for natural product based pharmaceuticals. Of these, lysine (Lys)-derived alkaloids exhibit a wide range of pharmacological properties which are beneficial for humans. For instance they have anticancer, anti-Alzheimer's disease, anti-inflammatory, hypocholesterolemic and antiarrhtymic effects. Lys-derived alkaloids are widely distributed throughout the plant kingdom: they can be found in various species from clubmosses to flowering plants. Lys is one of the most essential amino acids for humans and livestock and is synthesized in the plastids of land plants. Lys-derived alkaloids can be divided into four major groups including quinolizidine, lycopodium, piperidine, and indolizidine alkaloids. Despite the importance of these compounds, the biosynthetic pathways of Lys-derived alkaloids are not well understood. With the exception of indolizidine alkaloids, Lys decarboxylase (LDC) is the enzyme involved in the first committed step of the biosynthesis by catalyzing the transformation of L-Lys into cadaverine. Cadaverine is then oxidized by copper amine oxidase (CuAO) and spontaneously cyclized to Δ1-piperideine Schiff base which is a universal intermediate for the production of various Lys-derived alkaloids. CONCLUSION: In this review, we briefly summarize the recent understanding about the structures, occurrences, analytical procedures, biosyntheses, and potential health effects and medical applications of Lys-derived alkaloids with emphasis on quinolizidine alkaloids (QAs).

Onocerin Biosynthesis Requires Two Highly Dedicated Triterpene Cyclases in a Fern Lycopodium clavatum.

Onocerin is known for its unusual structure among triterpenoids, with a symmetrical structure that is formed by cyclizations at the both termini of dioxidosqualene. The nature of the enzyme catalyzing these unusual cyclizations has remained elusive for decades. Here, we report the cloning of genes responsible for these reactions; they exhibited unprecedented substrate specificities among oxidosqualene cyclase family members. Two genes, LCC and LCD, were identified from the fern Lycopodium clavatum. Expression in yeast revealed that both were required to produce α-onocerin. LCC, the first dioxidosqualene cyclase, catalyzed the production of a novel intermediate pre-α-onocerin from only dioxidosqualene as a substrate; LCD catalyzed the second half of the cyclization, exclusively from pre-α-onocerin. These results demonstrated that these two most unusual oxidosqualene cyclases were involved in onocerin biosynthesis.

Light-dependent reversible phosphorylation of the minor photosystem II antenna Lhcb6 (CP24) occurs in lycophytes.

Evolution of vascular plants required compromise between photosynthesis and photodamage. We analyzed representative species from two divergent lineages of vascular plants, lycophytes and euphyllophytes, with respect to the response of their photosynthesis and light-harvesting properties to increasing light intensity. In the two analyzed lycophytes, Selaginella martensii and Lycopodium squarrosum, the medium phase of non-photochemical quenching relaxation increased under high light compared to euphyllophytes. This was thought to be associated with the occurrence of a further thylakoid phosphoprotein in both lycophytes, in addition to D2, CP43 and Lhcb1-2. This protein, which showed light intensity-dependent reversible phosphorylation, was identified in S. martensii as Lhcb6, a minor LHCII antenna subunit of PSII. Lhcb6 is known to have evolved in the context of land colonization. In S. martensii, Lhcb6 was detected as a component of the free LHCII assemblies, but also associated with PSI. Most of the light-induced changes affected the amount and phosphorylation of the LHCII assemblies, which possibly mediate PSI-PSII connectivity. We propose that Lhcb6 is involved in light energy management in lycophytes, participating in energy balance between PSI and PSII through a unique reversible phosphorylation, not yet observed in other land plants.

Heathcock-inspired strategies for the synthesis of fawcettimine-type Lycopodium alkaloids.

The fawcettimine-type Lycopodium alkaloids have garnered significant attention from synthetic organic chemists since the isolation of fawcettimine in 1959. Despite being targets of interest for over 50 years, most of the strategies employed in the syntheses of fawcettimine congeners have built upon Inubushi and Heathcock's original work, realized in 1979 and 1986, respectively. This elegant strategy has been explored and expanded upon in the intervening years since the original publications, in what we now call the Heathcock-inspired strategy. While other disconnections have been disclosed, this strategy remains one of the most efficient. In this Concept article, we focus on exploring a number of recent Heathcock-inspired syntheses of fawcettimine-type Lycopodium alkaloids. We also briefly discuss alternative, novel disconnections.

Enhanced bioavailability of eicosapentaenoic acid from fish oil after encapsulation within plant spore exines as microcapsules.

Benefits of eicosapentaenoic acid (EPA) can be enhanced by raising their bioavailability through microencapsulation. Pollen can be emptied to form hollow shells, known as exines, and then used to encapsulate material, such as oils in a dry powder form. Six healthy volunteers ingested 4.6 g of fish oil containing 20% EPA in the form of ethyl ester first alone and then as 1:1 microencapsulated powder of exines and fish oil. Serum bioavailability of EPA was measured by area under curve (AUC(0-24)). The mean AUC(0-24) of EPA from ethyl ester with exine (M = 19.7, SD = 4.3) was significantly higher than ethyl ester without exines (M = 2, SD = 1.4, p < 0.01).The bioavailability of EPA is enhanced by encapsulation by pollen exines.

Total synthesis of (+)-complanadine A using an iridium-catalyzed pyridine C-H functionalization.

The total synthesis of the Lycopodium alkaloid complanadine A, which is an unsymmetrical dimer of lycodine, was achieved by exploiting a common tetracyclic precursor. Key to the success of the synthesis was the development of a late-stage site-selective C-H functionalization of a pyridine moiety to arrive at a key boronic ester intermediate.

Synthesis of (+)-complanadine A, an inducer of neurotrophic factor excretion.

A total synthesis of the Lycopodium alkaloid (+)-complanadine A is described. Complanadine A has been shown to induce the secretion of neurotrophic factors from 1321N1 cells, promoting the differentiation of PC-12 cells. The use of a simplifying metal mediated [2+2+2] + [2+2+2] sequence using a silyl-substituted diyne and 2 equiv of the corresponding alkyne-nitrile has provided rapid access to the natural product.

Causes and consequences of high osmotic potentials in epiphytic higher plants.

Past reports of the water relations of epiphytes, particularly bromeliads, indicate that tissue osmotic potentials in these tropical and subtropical plants are very high (close to zero) and are similar to values for aquatic plants. This is puzzling because several ecophysiological studies have revealed a high degree of drought stress tolerance in some of these epiphytes. The goal of this study was two-fold: (1) to increase the number of epiphytic taxa sampled for tissue osmotic potentials; and (2) to explain the apparent discrepancy in the significance of the tissue water relations and tolerance of drought stress in epiphytes. Tissue osmotic potentials of 30 species of epiphytic ferns, lycophytes, and orchids were measured in a subtropical rain forest in northeastern Taiwan. Nearly all values were less negative than -1.0 MPa, in line with all previous data for epiphytes. It is argued that such high osmotic potentials, indicative of low solute concentrations, are the result of environmental constraints of the epiphytic habitat on productivity of these plants, and that low rates of photosynthesis and transpiration delay the onset of turgor loss in the tissues of epiphytes such that they appear to be very drought-stress tolerant. Maintenance of photosynthetic activity long into drought periods is ascribed to low rates of transpiration and, hence, delayed tissue desiccation, and hydration of the photosynthetic tissue at the expense of water from the water-storage parenchyma.