Cyclase Research Articles

Structure of epi-isozizaene synthase from Streptomyces coelicolor A3(2), a platform for new terpenoid cyclization templates.

The X-ray crystal structure of recombinant epi-isozizaene synthase (EIZS), a sesquiterpene cyclase from Streptomyces coelicolor A3(2), has been determined at 1.60 A resolution. Specifically, the structure of wild-type EIZS is that of its closed conformation in complex with three Mg(2+) ions, inorganic pyrophosphate (PP(i)), and the benzyltriethylammonium cation (BTAC). Additionally, the structure of D99N EIZS has been determined in an open, ligand-free conformation at 1.90 A resolution. Comparison of these two structures provides the first view of conformational changes required for substrate binding and catalysis in a bacterial terpenoid cyclase. Moreover, the binding interactions of BTAC may mimic those of a carbocation intermediate in catalysis. Accordingly, the aromatic rings of F95, F96, and F198 appear to be well-oriented to stabilize carbocation intermediates in the cyclization cascade through cation-pi interactions. Mutagenesis of aromatic residues in the enzyme active site results in the production of alternative sesquiterpene product arrays due to altered modes of stabilization of carbocation intermediates as well as altered templates for the cyclization of farnesyl diphosphate. Accordingly, the 1.64 A resolution crystal structure of F198A EIZS in a complex with three Mg(2+) ions, PP(i), and BTAC reveals an alternative binding orientation of BTAC; alternative binding orientations of a carbocation intermediate could lead to the formation of alternative products. Finally, the crystal structure of wild-type EIZS in a complex with four Hg(2+) ions has been determined at 1.90 A resolution, showing that metal binding triggers a significant conformational change of helix G to cap the active site.

Diagnosis and treatment of guanylyl cyclase-C and colorectal cancer

Guanylyl cyelase-C (GC-C) is recently reported as the intestinal specific polypeptide receptor,which is expressed selectively by cells from intestinal mucosa,including normal intestinal cells,primary and metastatic colorectal cancer cells.Stimulated by heat-stable enterotoxin (STa),guanylin and uroguanylin,GC-C could not only regulate the proliferation and differentiation of intestinal cells,but also inhibit the proliferation of colorectal cancer cells and DNA synthesis.Micrometastases of the colorectal cancer can be found in lymph nodes,peripheral blood and liver lobes by GC-C detection.We can also perform GC-C and ligands for targeted prevention and therapy of colorectal cancer.GCC play important roles in detecting occult micrometastasos of colorectal cancer and targeted therapy. Key words: Guanylate cyclase; Colorectal neoplasms; Neoplasm metastasis; Diagnosis; Therapy

The protective effect of pituitary adenylate cyclase activating polypeptide on ischemic neuronal damage

垂体腺苷酸环化酶激活肽(PACAP)是一种新发现的神经肽,具有多种生物学活性.近年来PACAP的神经保护和神经营养作用越来越引起人们的注意.在缺血性脑损伤中,它能减少兴奋性氨基酸的释放,清除自由基,抑制炎性细胞趋化及神经元和小脑颗粒细胞的凋亡等,且PACAP相对分子质量小,能完全通过血脑屏障,为临床治疗缺血性脑损伤提供新的途径。

Crystal Structure of (+)-δ-Cadinene Synthase from Gossypium arboreum and Evolutionary Divergence of Metal Binding Motifs for Catalysis

(+)-Delta-cadinene synthase (DCS) from Gossypium arboreum (tree cotton) is a sesquiterpene cyclase that catalyzes the cyclization of farnesyl diphosphate in the first committed step of the biosynthesis of gossypol, a phytoalexin that defends the plant from bacterial and fungal pathogens. Here, we report the X-ray crystal structure of unliganded DCS at 2.4 A resolution and the structure of its complex with three putative Mg(2+) ions and the substrate analogue inhibitor 2-fluorofarnesyl diphosphate (2F-FPP) at 2.75 A resolution. These structures illuminate unusual features that accommodate the trinuclear metal cluster required for substrate binding and catalysis. Like other terpenoid cyclases, DCS contains a characteristic aspartate-rich D(307)DTYD(311) motif on helix D that interacts with Mg(2+)(A) and Mg(2+)(C). However, DCS appears to be unique among terpenoid cyclases in that it does not contain the "NSE/DTE" motif on helix H that specifically chelates Mg(2+)(B), which is usually found as the signature sequence (N,D)D(L,I,V)X(S,T)XXXE (boldface indicates Mg(2+)(B) ligands). Instead, DCS contains a second aspartate-rich motif, D(451)DVAE(455), that interacts with Mg(2+)(B). In this regard, DCS is more similar to the isoprenoid chain elongation enzyme farnesyl diphosphate synthase, which also contains two aspartate-rich motifs, rather than the greater family of terpenoid cyclases. Nevertheless, the structure of the DCS-2F-FPP complex shows that the structure of the trinuclear magnesium cluster is generally similar to that of other terpenoid cyclases despite the alternative Mg(2+)(B) binding motif. Analyses of DCS mutants with alanine substitutions in the D(307)DTYD(311) and D(451)DVAE(455) segments reveal the contributions of these segments to catalysis.

SOC: now also store-operated cyclase

SOC: now also store-operated cyclase

Selective coupling of type 6 adenylyl cyclase with type 2 IP3receptors mediates direct sensitization of IP3receptors by cAMP

Tovey et al. 2008. J. Cell Biol. doi:10.1083/jcb.200803172 [OpenUrl][1][Abstract/FREE Full Text][2] [1]: {openurl}?query=rft_id%253Dinfo%253Adoi%252F10.1083%252Fjcb.200803172%26rft_id%253Dinfo%253Apmid%252F18936250%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%

Sporulenes, three new polycyclic terpenoids from Bacillus subtilis spores

Hopanoids are ubiquitous polycyclic isoprenoids derived from terpene metabolism. While hopanoids are believed to regulate membrane fluidity in prokaryotes, the significance of their structural diversity is not understood. Analysis of numerous bacterial species has revealed this class of compounds to be widespread among diverse taxonomic groups [1], but polycyclic terpenoids have yet to be detected in a large proportion of bacterial species. Previous investigations have failed to detect hopanoid-like compounds produced by Bacillus subtilis, for example, despite the presence of a putative squalene-hopene cyclase enzyme (SqhC) in its genome. By comparing lipid profiles of wild-type B. subtilis and an SqhC-deficient strain, we identified three novel tetracyclic terpenoids, the sporulenes, believed to be products of the B. subtilis SqhC. Structures of these compounds were deduced by NMR and mass spectroscopic methods. The unusual scalarane backbone of the sporulenes arises from cyclization of a regular heptaprenyl precursor rather than the more common substrate of terpenoid cyclases, squalene. The discovery of these monoaromatic terpenoids in bacterial spores highlights the structural diversity of terpenoids and underscores our modest understanding of their physiological functions.

Terpenoid cyclases: design and function of electrophilic catalysts.

Terpenoid cyclases catalyse the cyclization of the universal acyclic precursors geranyl and farnesyl diphosphate to monoterpenes and sesquiterpenes, respectively. All such cyclases investigated to date are operationally soluble, moderately lipophilic proteins of relative molecular weight 40,000-100,000, requiring no cofactors other than a divalent metal, usually Mg2+ and occasionally Mn2+. The focus of most work has been on the mechanisms of the cyclization reactions themselves. It is currently proposed that the cyclase binds the acyclic substrate in a suitable conformation and initiates the cyclization by ionization of the labile allylic diphosphate moiety. The use of stereospecifically labelled substrates and analysis of the sites of labelling in the derived cyclization products has allowed the proposal of detailed cyclization mechanisms. Further insight into the architecture and function of the cyclase active site has come from the study of substrate and intermediate analogues designed to act as potential inhibitors or anomalous substrates of the normal cyclization reaction. Progress has also been made on the cloning of the relevant structural genes for sesquiterpene cyclases. This has led to new insights into the basic requirements for cyclase catalysis and specificity.

BAY 58–2667, a soluble guanylate cyclase activator, improves cardiopulmonary haemodynamics in acute decompensated heart failure and has a favourable safety profile

Address: 1HELIOS-Klinikum Erfurt, 99089 Erfurt, Germany, 2Kerckhoff-Klinik Nauheim, 61231 Nauheim, Germany, 3Schuchtermann-Klinik Bad Rothenfelde, 49214 Bad Rothenfelde, Germany, 4St Johannes Hospital Dortmund,44137 Dortmund, Germany, 5Universitat Halle, 06097 Halle, Germany, 6HELIOS-Klinikum Wuppertal, 42283 Wuppertal, Germany, 7Clinical Pharmacology, Bayer HealthCare AG, Pharma Research Centre, 42096 Wuppertal, Germany and 8Global Biostatistics, Bayer HealthCare AG, Pharma Research Centre, 42096 Wuppertal, Germany

Exploring biosynthetic diversity with trichodiene synthase

Trichodiene synthase is a terpenoid cyclase that catalyzes the cyclization of farnesyl diphosphate (FPP) to form the bicyclic sesquiterpene hydrocarbon trichodiene (89%), at least five sesquiterpene side products (11%), and inorganic pyrophosphate (PP i). Incubation of trichodiene synthase with 2-fluorofarnesyl diphosphate or 4-methylfarnesyl diphosphate similarly yields sesquiterpene mixtures despite the electronic effects or steric bulk introduced by substrate derivatization. The versatility of the enzyme is also demonstrated in the 2.85 Å resolution X-ray crystal structure of the complex with Mg 2 + 3 - PP i and the benzyl triethylammonium cation, which is a bulkier mimic of the bisabolyl carbocation intermediate in catalysis. Taken together, these findings show that the active site of trichodiene synthase is sufficiently flexible to accommodate bulkier and electronically-diverse substrates and intermediates, which could indicate additional potential for the biosynthetic utility of this terpenoid cyclase.

Structure of limonene synthase, a simple model for terpenoid cyclase catalysis.

The crystal structure of (4S)-limonene synthase from Mentha spic ata, a metal ion-dependent monoterpene cyclase that catalyzes the coupled isomerization and cyclization of geranyl diphosphate, is reported at 2.7-A; resolution in two forms liganded to the substrate and intermediate analogs, 2-fluorogeranyl diphosphate and 2-fluorolinalyl diphosphate, respectively. The implications of these findings are described for domain interactions in the homodimer and for changes in diphosphate-metal ion coordination and substrate binding conformation in the course of the multistep reaction.

X-ray Crystal Structure of Aristolochene Synthase from Aspergillus terreus and Evolution of Templates for the Cyclization of Farnesyl Diphosphate,

Aristolochene synthase from Aspergillus terreus catalyzes the cyclization of the universal sesquiterpene precursor, farnesyl diphosphate, to form the bicyclic hydrocarbon aristolochene. The 2.2 A resolution X-ray crystal structure of aristolochene synthase reveals a tetrameric quaternary structure in which each subunit adopts the alpha-helical class I terpene synthase fold with the active site in the "open", solvent-exposed conformation. Intriguingly, the 2.15 A resolution crystal structure of the complex with Mg2+3-pyrophosphate reveals ligand binding only to tetramer subunit D, which is stabilized in the "closed" conformation required for catalysis. Tetramer assembly may hinder conformational changes required for the transition from the inactive open conformation to the active closed conformation, thereby accounting for the attenuation of catalytic activity with an increase in enzyme concentration. In both conformations, but especially in the closed conformation, the active site contour is highly complementary in shape to that of aristolochene, and a catalytic function is proposed for the pyrophosphate anion based on its orientation with regard to the presumed binding mode of aristolochene. A similar active site contour is conserved in aristolochene synthase from Penicillium roqueforti despite the substantial divergent evolution of these two enzymes, while strikingly different active site contours are found in the sesquiterpene cyclases 5-epi-aristolochene synthase and trichodiene synthase. Thus, the terpenoid cyclase active site plays a critical role as a template in binding the flexible polyisoprenoid substrate in the proper conformation for catalysis. Across the greater family of terpenoid cyclases, this template is highly evolvable within a conserved alpha-helical fold for the synthesis of terpene natural products of diverse structure and stereochemistry.

Cyclic nucleotide phosphodiesterase (PDE) inhibitors: novel therapeutic agents for progressive renal disease.

Cyclic nucleotides are recognized as critical mediators of many renal functions, including solute transport, regulation of vascular tone, proliferation of parenchymal cells, and inflammation. Although most studies have linked elevated cAMP levels to activation of protein kinase A, cAMP can also directly activate cyclic nucleotide gated ion channels and can signal through activation of GTP exchange factors. Cyclic AMP signaling is highly compartmentalized through plasma membrane localization of adenylyl cyclase and expression of scaffolding proteins that anchor protein kinase A to specific intracellular locations. Cyclic nucleotide levels are largely regulated through catabolic processes directed by phosphodiesterases (PDEs). The PDE superfamily is large and complex, with over 60 distinct isoforms that preferentially hydrolyze cAMP, cGMP, or both. PDEs contribute to compartmentalized cyclic nucleotide signaling. The unique cell- and tissue-specific distribution of PDEs has prompted the development of highly specific PDE inhibitors to treat a variety of inflammatory conditions. In experimental systems, PDE inhibitors have been employed to demonstrate functional compartmentalization of cyclic nucleotide signaling in the kidney. For example, mitogenesis in glomerular mesangial cells and normal tubular epithelial cells is negatively regulated by an intracellular pool of cAMP that is metabolized by PDE3, but not by other PDEs. In Madin-Darby canine kidney cells, an in vitro model of polycystic kidney disease, an intracellular pool of cAMP directed by PDE3 stimulates mitogenesis. In mesangial cells, an intracellular pool of cAMP directed by PDE4 inhibits reactive oxygen species and expression of the potent proin-flammatory cytokine monocyte chemoattractant protein 1. An intracellular pool of cGMP directed by PDE5 regulates solute transport. PDE5 inhibitors ameliorate renal injury in a chronic renal disease model. In this overview, we highlight recent studies to define relationships between PDE expression and renal function and to provide evidence that PDE inhibitors may be effective agents in treating chronic renal disease.

X‐ray Crystal Structure of Aristolochene Synthase from Aspergillus terreus and Evolution of Templates for the Cyclization of Farnesyl Diphosphate

Aristolochene synthase from Aspergillus terreus catalyzes the cyclization of the universal sesquiterpene precursor, farnesyl diphosphate, to form the bicyclic hydrocarbon aristolochene. The 2.2 Å resolution X-ray crystal structure of aristolochene synthase reveals a tetrameric quaternary structure in which each subunit adopts the α-helical class I terpene synthase fold with the active site in the “open”, solvent-exposed conformation. Intriguingly, the 2.15 Å resolution crystal structure of the complex with Mg2+3-pyrophosphate reveals ligand binding only to tetramer subunit D, which is stabilized in the “closed” conformation required for catalysis. In both conformations, but especially so in the closed conformation, the active site contour is highly complementary in shape to that of aristolochene, and a catalytic function is proposed for the pyrophosphate anion based on its orientation with regard to the presumed binding mode of aristolochene. A similar active site contour is conserved in aristolochene synthase from P. roqueforti despite the substantial divergent evolution of these two enzymes, while strikingly different active site contours are found in the sesquiterpene cyclases 5-epi aristolochene synthase and trichodiene synthase. Thus, the terpenoid cyclase active site plays a critical role as a template to bind the flexible polyisoprenoid substrate in the proper conformation for catalysis. NIH Grants GM 56838 (D.W.C.) and GM 30301 (D.E.C.)

Recent advances in cartilage metabolism research

The past few years have seen an enormous increase in the knowledge of the signaling pathways and the transcription factors that control endochondral bone development. Recent studies of rare chondrodysplasias that exhibit disproportionate dwarfism due to the impairment of endochondral ossification have identified many responsible genes and have clarified pathogenic mechanisms of these diseases by using molecular genetics and biology. The details of human diseases related to collagen type II alpha1 (COL2A1), collagen type X alpha1 (COL10A1), parathyroid hormone (PTH) /parathyroid hormone-related protein (PTHrP) receptor, guanylate cyclase B (GC-B), sry-box 9 (SOX9), runt-related transcription factor 2 (RUNX2), which play important roles in the process of endochondral ossification, will be outlined.

Histological function of PTHrP in cartilage

Parathyroid hormone-related peptide (PTHrP) is known as an important local factor for chondrogenesis, promoting chondrocyte proliferation and inhibiting their differentiation into the hypertrophic phenotype. Signaling transduction through the PTH/PTHrP receptor has two possible pathways: the activation of adenylate cyclase and subsequent protein kinase A (PKA), and the activation of phospholipase C (PLC). Recent studies with mice carrying PTH/PTHrP receptor inactivated for PLC and chondrocyte-specific deletion of the G (s) gene have shown that cAMP/PKA signaling appears to stimulate chondrocyte proliferation and inhibit their differentiation, whereas PLC signaling enhanced chondrocyte differentiation and inhibited their proliferation. In a physiological state, cAMP/PKA signaling may predominate over PLC pathway. Also, Na(+)/H(+)exchanger regulatory factor 2 (NHERF2) has been reported to down-regulate adenylate cyclase activity, in a switch mechanism that results in signal transduction through the PLC pathway.

Different distribution of nitric oxide synthase and soluble guanylyl cyclase activities in the detrusor and proximal urethra of the rabbit.

We investigated the different distributions of nitric oxide synthase (NOS) and soluble guanylate cyclase activities in the detrusor and proximal urethra in the rabbit. The detrusor and proximal urethra were excised, the mucosa and muscularis were separated and each layer was homogenized to measure the activities of NOS as well as baseline and sodium nitroprusside (SNP) activated soluble guanylate cyclase. Isometric tension experiments were performed in detrusor and urethral strips with and without mucosa. In the detrusor NOS and sodium nitroprusside activated soluble guanylate cyclase activities in the mucosa were much higher than in the muscularis. While in the proximal urethra NOS activity in the 2 layers was similar, SNP activated soluble guanylate cyclase activity in the muscularis was 2.8 times higher than in the mucosa. Removing the mucosa reduced nitric oxide (NO) mediated neurogenic relaxation in the proximal urethra, while no relaxation responses could be elicited by electrical field stimulation with or without mucosa in the detrusor. On the other hand, removing the mucosa did not affect SNP or 8-bromo-cyclic guanosine monophosphate induced relaxation in the detrusor and proximal urethra. In the detrusor NOS and soluble guanylate cyclase activities were mainly detected in the mucosa, while in the urethra these activities were distributed throughout the mucosa and muscularis. Also, the mucosa in the urethra but not in the detrusor contributed to electrical field stimulation induced, NO mediated relaxation. These findings confirm previous studies demonstrating that NO has a different role in the detrusor than in the proximal urethra.

Neuropeptides and retinal development.

Different peptidergic systems have been investigated with some detail during retinal development, including substance P (SP), vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase activating polypeptide (PACAP) and somatostatin (SRIF). Concerning possible developmental actions of neuropeptides, VIP and PACAP exert protective and growth-promoting actions that may sustain retinal neurons during their development. In addition, the presence of transient SRIF expressing cells and recent observations in SRIF receptor knock out mice indicate variegated roles of this peptide in the development of the retina and of retinofugal projections. Finally, recent studies have shown that, in the developing rabbit retina, changes in the expression pattern of SP receptors are accompanied by modifications of SP physiological effects, indicating that retinal circuits where SP is involved are likely to function in a substantially different manner before the retina becomes involved in the processing of visual stimuli. SP neurotransmission in the immature retina may subserve developmental events, and SP is likely to represent an important developmental factor for the maturation of retinal neurons and circuitries.

Nitric oxide and platelet energy metabolism.

This study was undertaken to determine whether nitric oxide (NO) can affect platelet responses through the inhibition of energy production. It was found that NO donors: S-nitroso-N-acetylpenicyllamine, SNAP, (5-50 microM) and sodium nitroprusside, SNP, (5-100 microM) inhibited collagen- and ADP-induced aggregation of porcine platelets. The corresponding IC50 values for SNAP and SNP varied from 5 to 30 microM and from 9 to 75 microM, respectively. Collagen- and thrombin-induced platelet secretion was inhibited by SNAP (IC50 = 50 microM) and by SNP (IC50 = 100 microM). SNAP (20-100 microM), SNP (10-200 microM) and collagen (20 microg/ml) stimulated glycolysis in intact platelets. The degree of glycolysis stimulation exerted by NO donors was similar to that produced by respiratory chain inhibitors (cyanide and antimycin A) or uncouplers (2,4-dinitrophenol). Neither the NO donors nor the respiratory chain blockers affected glycolysis in platelet homogenate. SNAP (20-100 microM) and SNP (50-200 microM) inhibited oxygen consumption by platelets. The effect of SNP and SNAP on glycolysis and respiration was not reduced by 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one, a selective inhibitor of NO-stimulated guanylate cyclase. SNAP (5-100 microM) and SNP (10-300 microM) inhibited the activity of platelet cytochrome oxidase and had no effect on NADH:ubiquinone oxidoreductase and succinate dehydrogenase. Blocking of the mitochondrial energy production by antimycin A slightly affected collagen-evoked aggregation and strongly inhibited platelet secretion. The results indicate that: 1) in porcine platelets NO is able to diminish mitochondrial energy production through the inhibition of cytochrome oxidase, 2) the inhibitory effect of NO on platelet secretion (but not aggregation) can be attributed to the reduction of mitochondrial energy production.

Vitis vinifera terpenoid cyclases: functional identification of two sesquiterpene synthase cDNAs encoding (+)-valencene synthase and (−)-germacrene D synthase and expression of mono- and sesquiterpene synthases in grapevine flowers and berries

Valencene is a volatile sesquiterpene emitted from flowers of grapevine, Vitis vinifera L. A full-length cDNA from the cultivar Gewürztraminer was functionally expressed in Escherichia coli and found to encode valencene synthase (VvVal). The two major products formed by recombinant VvVal enzyme activity with farnesyl diphosphate (FPP) as substrate are (+)-valencene and (−)-7- epi-α-selinene. Grapevine valencene synthase is closely related to a second sesquiterpene synthase from this species, (−)-germacrene D synthase ( VvGerD). VvVal and VvGerD cDNA probes revealed strong signals in Northern hybridizations with RNA isolated from grapevine flower buds. Transcript levels were lower in open pre-anthesis flowers, flowers after anthesis, or at early onset of fruit development. Similar results were obtained using a third probe, (−)-α-terpineol synthase, a monoterpenol synthase. Sesquiterpene synthase and monoterpene synthase transcripts were not detected in the mesocarp and exocarp during early stages of fruit development, but transcripts hybridizing with VvVal appeared during late ripening of the berries. Sesquiterpene synthase transcripts were also detected in young seeds.