Showing papers on "Cyclase published in 2000"
TL;DR: Membrane-bound P2-receptors mediate the actions of extracellular nucleotides in cell-to-cell signalling and may be involved in the modulation of neuro-neural signalling transmission.
Abstract: Membrane-bound P2-receptors mediate the actions of extracellular nucleotides in cell-to-cell signalling. P2X-receptors are ligand-gated ion channels, whereas P2Y-receptors belong to the superfamily of G-protein-coupled receptors. So far, the P2Y family is composed of eight cloned and functionally defined subtypes. Five of them (P2Y1, P2Y2, P2Y4, P2Y6 and P2Y11) are present in human tissues. The P2Y3-, p2y8- and tp2y-receptors may be species orthologues. The principal physiological agonists of the cloned human P2Y-receptors are ADP (P2Y1), UTP/ATP (P2Y2), UTP (P2Y4), UDP (P2Y6) and ATP (P2Y11). The rat P2Y4-receptor is activated by both UTP and ATP. Specific patterns of polar amino acid residues in the exofacial portions of transmembrane domains (TMs) 6 and 7 of the P2Y-receptors may account for the ligand specificity of the subtypes. Suramin acts as an antagonist at most P2Y-receptors with the exception of P2Y4- and tp2y-receptors. PPADS has been shown to block P2Y1-, the human P2Y4- and P2Y6-receptors. The nucleotide analogue 2'-deoxy-N6-methyladenosine-3',5'-bisphosphate (MRS 2179), in contrast, seems to be a potent and selective antagonist at the P2Y1-receptor. All cloned and functionally expressed P2Y-receptors are able to couple to phospholipase C. The P2Y11-receptor mediates in addition a stimulation of adenylate cyclase and the tp2y-receptor an inhibition of this signal transduction pathway. Other functionally defined subtypes, e.g., the receptor mediating an inhibition of adenylate cyclase in blood platelets, are not yet cloned.
510 citations
TL;DR: It is shown that NF1 affects learning and short-term memory independently of its developmental effects, and that the mechanism of the NF1-dependent activation of the Rut-adenylyl cyclase pathway is essential for mediating Drosophila learning and memory.
Abstract: The tumour-suppressor gene Neurofibromatosis 1 (Nf1) encodes a Ras-specific GTPase activating protein (Ras-GAP)1,2,3,4,5. In addition to being involved in tumour formation6,7, NF1 has been reported to cause learning defects in humans8,9,10 and Nf1 knockout mice11. However, it remains to be determined whether the observed learning defect is secondary to abnormal development. The Drosophila NF1 protein is highly conserved, showing 60% identity of its 2,803 amino acids with human NF1 (ref. 12). Previous studies have suggested that Drosophila NF1 acts not only as a Ras-GAP but also as a possible regulator of the cAMP pathway that involves the rutabaga (rut)-encoded adenylyl cyclase13. Because rut was isolated as a learning and short-term memory mutant14,15, we have pursued the hypothesis that NF1 may affect learning through its control of the Rut-adenylyl cyclase/cAMP pathway. Here we show that NF1 affects learning and short-term memory independently of its developmental effects. We show that G-protein-activated adenylyl cyclase activity consists of NF1-independent and NF1-dependent components, and that the mechanism of the NF1-dependent activation of the Rut-adenylyl cyclase pathway is essential for mediating Drosophila learning and memory.
255 citations
TL;DR: Structural homology of this fungal cyclase with plant and bacterial terpenoid cyclases, despite minimal amino acid sequence identity, suggests divergence from a common, primordial ancestor in the evolution of terpene biosynthesis.
165 citations
TL;DR: In tobacco leaves that transiently express T.NSY, an increase in neoxanthin content with a concomitant decrease in violaxanthin is observed, suggesting that these two enzymes were remodeled from LCY during higher plant evolution to create new forms of oxidized carotenoids.
Abstract: Neoxanthin, a precursor of the plant hormone abscisic acid, is an allenic xanthophyll recognized as the last product of carotenoid synthesis in green plants. A cDNA for neoxanthin synthase (NSY) was isolated from tomato using a molecular approach based on the mechanistic and structural similarities of NSY to two other closely related carotenogenic enzymes, lycopene cyclase (LCY) and capsanthin-capsorubin synthase (CCS). The identified tomato NSY cDNA (T.NSY) encodes a 56-kDa plastid-targeted protein that when expressed in Escherichia coli, catalyzes the conversion of violaxanthin to neoxanthin. In tobacco leaves that transiently express T.NSY, an increase in neoxanthin content with a concomitant decrease in violaxanthin is observed. NSY is structurally similar to LCY and CCS. However, in Cyanobacteria, the generally accepted progenitor of plastids, both CCS and NSY are absent while LCY is present. LCY catalyzes a simplified version of the reaction catalyzed by NSY and CCS suggesting that these two enzymes were remodeled from LCY during higher plant evolution to create new forms of oxidized carotenoids.
163 citations
TL;DR: It is shown here that an activated allele of gpa2 (gpa2(R176H), carrying a mutation in the coding region for the GTPase domain) fully suppresses mutations in git3 and git5, leading to a refinement in the model.
Abstract: The fission yeast Schizosaccharomyces pombe responds to environmental glucose by activating adenylate cyclase. The resulting cAMP signal activates protein kinase A (PKA). PKA inhibits glucose starvation-induced processes, such as conjugation and meiosis, and the transcription of the fbp1 gene that encodes the gluconeogenic enzyme fructose-1,6-bisphosphatase. We previously identified a collection of git genes required for glucose repression of fbp1 transcription, including pka1/git6, encoding the PKA catalytic subunit, git2/cyr1, encoding adenylate cyclase, and six "upstream" genes required for adenylate cyclase activation. The git8 gene, identical to gpa2, encodes the alpha subunit of a heterotrimeric guanine-nucleotide binding protein (Galpha) while git5 encodes a Gbeta subunit. Multicopy suppression studies with gpa2(+) previously indicated that S. pombe adenylate cyclase activation may resemble that of the mammalian type II enzyme with sequential activation by Galpha followed by Gbetagamma. We show here that an activated allele of gpa2 (gpa2(R176H), carrying a mutation in the coding region for the GTPase domain) fully suppresses mutations in git3 and git5, leading to a refinement in our model. We describe the cloning of git3 and show that it encodes a putative seven-transmembrane G protein-coupled receptor. A git3 deletion confers the same phenotypes as deletions of other components of the PKA pathway, including a germination delay, constitutive fbp1 transcription, and starvation-independent conjugation. Since the git3 deletion is fully suppressed by the gpa2(R176H) allele with respect to fbp1 transcription, git3 appears to encode a G protein-coupled glucose receptor responsible for adenylate cyclase activation in S. pombe.
128 citations
TL;DR: A strong structural homology between the active sites of CD38 and the Aplysiacyclase is indicated, which is consistent with the two tryptophans serving a substrate positioning function.
128 citations
TL;DR: The pseudomature form of taxadiene synthase having 60 amino acids deleted from the preprotein was found to be superior with respect to level of expression, ease of purification, solubility, stability, and catalytic activity with kinetics comparable to the native enzyme.
105 citations
TL;DR: Based on the in vitro and in vivo results, the organization of the phytoene desaturase from N. crassa was proposed as an assembly of identical protein units which are responsible for the multistep reaction, but the spatial arrangement should be loose enough to allow an exchange of individual intermediates in both directions in and out of this complex.
100 citations
TL;DR: It is confirmed that adenylate cyclase activation by forskolin results in diminishedO-GlcNAc modification of several cellular proteins which can be overcome by exposure of the cells to glucosamine but not glucose, suggesting the PKA activation results in depletion of UDP-GlCNAc for O-glycosylation.
89 citations
TL;DR: Results suggest that PGE2 directly inhibits bone-resorbing activity of functionally mature osteoclasts by activation of the adenylate cyclase system, perhaps mainly through EP4.
Abstract: Prostaglandins (PGs) are well known to be important local factors in regulating bone formation and resorption. PGE2 is a potent stimulator of bone resorption because of enhancing osteoclast formation by its indirect action through stromal cells. However, the direct action of PGE2 on functionally mature osteoclasts is still controversial. In this study using highly purified rabbit mature osteoclasts, we examined the direct effect of PGE2 on osteoclastic bone-resorbing activity and its mechanism. PGE2 inhibited resorption pit formation on a dentine slice by the purified osteoclasts in a dose- and time-dependent manner. The inhibitory effect appeared as early as 4 hours after the PGE2 addition. Forskolin and 12-0-tetradecanoyl phorbol-13-acetate (TPA), respective activators of adenylate cyclase and protein kinase C, also decreased the osteoclastic bone-resorbing activity. PGE2 increased the content of intracellular cAMP in a dose range effective for the inhibition of bone resorption, whereas the prostanoid did not alter the intracellular level of inositol triphosphate. The inhibition of osteoclastic bone resorption by PGE2 was amplified and diminished by a cAMP phosphodiesterase inhibitor (isobutyl methylxanthine) and a protein kinase A inhibitor (Rp-cAMP), respectively. Of four different subtypes of PGE2 receptors (EPs), EP4 mRNA was predominantly expressed in isolated osteoclasts, whereas the other types of EP mRNA were detected in only small amounts. These results suggest that the PGE2 inhibitory effect was mediated by an adenylate cyclase system coupled with EP4. This possible association of PGE2 with EP4 in mature osteoclasts was supported by the finding that a specific agonist of EP4 (AE-604) inhibited the bone-resorbing activity and elevated the intracellular cAMP content. However, butaprost, a selective EP2 agonist, also mimicked the PGE2 effects on isolated osteoclasts although EP2 mRNA expression was minimal. In conclusion, PGE2 directly inhibits bone-resorbing activity of functionally mature osteoclasts by activation of the adenylate cyclase system, perhaps mainly through EP4.
77 citations
TL;DR: The nucleotide sequence of a full-length cDNA encoding ent-kaurene synthase that was isolated by a reverse-transcription polymerase chain reaction from Gibberella fujikuroi (Gcps/ks) is reported, suggesting that GcPS/ks is also a bifunctional diterpene cyclase.
Abstract: We report here the nucleotide sequence of a full-length cDNA encoding ent-kaurene synthase that was isolated by a reverse-transcription polymerase chain reaction from Gibberella fujikuroi (Gcps/ks). This cDNA encodes 952 amino acid residues with a relative molecular mass of 107 kDa. The sequence similarity between Gcps/ks and ent-kaurene synthase of the gibberellin A1-producing fungus, Phaeosphaeria sp. L487, is very high, suggesting that Gcps/ks is also a bifunctional diterpene cyclase. Its recombinant protein expressed in Escherichia coli converted geranylgeranyl diphosphate to copalyl diphosphate and ent-kaurene.
TL;DR: Kinetic analysis and identification of the two cyclase domains in a bifunctional diterpene cyclase, Phaeosphaeria ent-kaurene synthase (FCPS/KS) indicate that there are two separate interacting domains in the 106-kDa polypeptide of FCPS/ KS.
TL;DR: The results suggest that the interaction of two classes of ecto-ADP-ribose transfer enzymes plays an important role in immune regulation by the selective induction of apoptosis in activated T cells and that cADPR mediated signaling is essential for the survival ofactivated T cells.
TL;DR: Calcium feedback in vertebrate photoreceptors regulates synthesis of cGMP, a second messenger in phototransduction, and contributes to photoreceptor recovery and light adaptation.
TL;DR: Changes in the cardiac beta -adrenoceptor-G-protein(s)-adenylyl cyclase system occur that resemble those observed in human primary pulmonary hypertension, and MCT-treated rat appears to be a suitable animal model to study in more detail the pathophysiology of the development of right heart failure, and to identify new therapeutic possibilities.
TL;DR: A possible role for cADPR as an endogenous modulator of [Ca(2+)](i) in porcine TSM cells is suggested.
TL;DR: The data suggest that Gbeta is required for activation of adenylate cyclase either by promoting the activation of Galpha or by independently activatingAdenylates cyclase subsequent to Galpha stimulation as seen in type II mammalian adenYLate Cyclase activation.
Abstract: Fission yeast adenylate cyclase is activated by the gpa2 Gα subunit of a heterotrimeric guanine-nucleotide binding protein (G protein). We show that the git5 gene, also required for this activation, encodes a Gβ subunit. In contrast to another study, we show that git5 is not a negative regulator of the gpa1 Gα involved in the pheromone response pathway. While 43% identical to mammalian Gβ's, the git5 protein lacks the amino-terminal coiled-coil found in other Gβ subunits, yet the gene possesses some of the coding capacity for this structure 5′ to its ORF. Although both gpa2 (Gα) and git5 (Gβ) are required for adenylate cyclase activation, only gpa2 is needed to maintain basal cAMP levels. Strains bearing a git5 disruption are derepressed for fbp1 transcription and sexual development even while growing in a glucose-rich environment, although fbp1 derepression is half that observed in gpa2 deletion strains. Multicopy gpa2 partially suppresses the loss of git5 , while the converse is not true. These data suggest that Gβ is required for activation of adenylate cyclase either by promoting the activation of Gα or by independently activating adenylate cyclase subsequent to Gα stimulation as seen in type II mammalian adenylate cyclase activation.
TL;DR: Two factors with major impact on the lifetime of NO-sGC are the proximity to NO scavengers and the calcium concentration in the cell, which suggest that both activation and deactivation are fast.
Abstract: Soluble guanylate cyclase (sGC) is highly activated by nitric oxide (NO) and is the known mediator of the effects of NO on a variety of physiological processes. The rates at which sGC is activated and deactivated are therefore of wide interest since they determine the duration of a tissue's response to NO. The effect of NO on smooth muscle dissipates in 1-2 min, suggesting that both activation and deactivation are fast. In vitro measurements show that the activation of sGC occurs in less than a second, while the deactivation takes several hours at 20 degrees C. However, recent reports indicate that Mg-GTP, oxyhemoglobin, and reducing and oxidizing agents could deactivate the cyclase in several seconds to minutes, though the effectiveness of each of these agents is in dispute. We investigated the lifetime of NO-sGC in the cytosol of retina by monitoring its enzymatic activity at 20 degrees C. Our results show that Mg-GTP, the substrate of NO-sGC, has no influence on the deactivation. Similarly, reducing agents glutathione and dithiothreitol shortened the half-life of NO-sGC only by about 30%. The greatest effect on the deactivation was caused by scavengers of NO: oxyhemoglobin reduced the half-life of NO-sGC from 106 min to 18 s; another NO scavenger, 2-(4-carboxyphenyl)-4,4,5, 5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO), reduced it to 42 s (20 degrees C). Similarly rapid deactivation was observed with the enzyme from bovine lung, immunoprecipitated enzyme from bovine retina, and heme-deficient enzyme from bovine retina reconstituted with heme. On the other hand, YC-1, an activator of sGC, stabilized the activated enzyme, preventing NO dissociation, as was evident from the inability of oxyhemoglobin or CPTIO to deactivate NO-sGC. Calcium, which is known to inhibit NO-sGC, also inhibited the effects of oxyhemoglobin and CPTIO, slowing down the deactivation of the enzyme. Lithium, which is also known to inhibit NO-sGC, had no effect on the deactivation rate of the enzyme. These results, taken together, suggest that two factors with major impact on the lifetime of NO-sGC are the proximity to NO scavengers and the calcium concentration in the cell.
TL;DR: Initiation and substrate selectivity may be determined by the interaction of the DDTAVV motif with the isopropylidene of squalene and of the DCTAEA motifwith the epoxide of oxidosqualene (for SHC and OSC), the first report of a substrate switch determined by a central catalytic motif in a triterpenoid cyclase.
TL;DR: The results demonstrate the existence of a signalling pathway from Ang II receptors to membrane-bound ADP-ribosyl cyclase in the ventricular muscle cell and suggest that the Ang II-induced increase in cADp-ribose synthesis is involved in the regulation of cardiac function and development.
Abstract: To examine the role of cyclic ADP-ribose (cADP-ribose) as a second messenger downstream of angiotensin II (Ang II) receptor activation in the heart, ADP-ribosyl cyclase activity was measured in a crude membrane fraction of ventricular myocytes. Ang II at 10-100 nM increased ADP-ribosyl cyclase activity by 40-90% in the ventricular muscle of neonatal (2-4-day-old) rats, but not in fetal or adult hearts. This increase was inhibited by the Ang II antipeptide. Stimulation of ADP-ribosyl cyclase was reproduced by GTP and guanosine 5'-[gamma-thio]triphosphate, and prevented by guanosine 5'-[beta-thio]diphosphate. Prior treatment of the rats with cholera toxin A and B subunits also blocked the Ang II-induced activation. The density of Ang II receptors detected as [(3)H]Ang II binding was higher in neonatal than adult rats. These results demonstrate the existence of a signalling pathway from Ang II receptors to membrane-bound ADP-ribosyl cyclase in the ventricular muscle cell and suggest that the Ang II-induced increase in cADP-ribose synthesis is involved in the regulation of cardiac function and development.
TL;DR: A 'partitioning' reaction scheme for the Aplysia enzyme is proposed, similar to that established previously for mammalian CD38/NAD(+) glycohydrolases, which involves the formation of a single oxocarbenium-type intermediate that partitions to cADP-ribose and solvolytic products via competing pathways.
Abstract: Highly purified Aplysia californica ADP-ribosyl cyclase was found to be a multifunctional enzyme. In addition to the known transformation of NAD(+) into cADP-ribose this enzyme is able to catalyse the solvolysis (hydrolysis and methanolysis) of cADP-ribose. This cADP-ribose hydrolase activity, which becomes detectable only at high concentrations of the enzyme, is amplified with analogues such as pyridine adenine dinucleotide, in which the cleavage rate of the pyridinium-ribose bond is much reduced compared with NAD(+). Although the specificity ratio V(max)/K(m) is in favour of NAD(+) by 4 orders of magnitude, this multifunctionality allowed us to propose a 'partitioning' reaction scheme for the Aplysia enzyme, similar to that established previously for mammalian CD38/NAD(+) glycohydrolases. This mechanism involves the formation of a single oxocarbenium-type intermediate that partitions to cADP-ribose and solvolytic products via competing pathways. In favour of this mechanism was the finding that the enzyme also catalysed the hydrolysis of NMN(+), a substrate that cannot undergo cyclization. The major difference between the mammalian and the invertebrate enzymes resides in their relative cyclization/hydrolysis rate-constant ratios, which dictate their respective yields of cADP-ribose (ADP-ribosyl cyclase activity) and ADP-ribose (NAD(+) glycohydrolase activity). For the Aplysia enzyme's catalysed transformation of NAD(+) we favour a mechanism where the formation of cADP-ribose precedes that of ADP-ribose; i.e. macroscopically the invertebrate ADP-ribosyl cyclase conforms to a sequential reaction pathway as a limiting form of the partitioning mechanism.
TL;DR: Site-directed mutagenesis indicated that Arg416 and Ser417 are essential for G protein activation and the proximal C terminus mediates signal transduction, and the distal is involved with internalization.
TL;DR: It is concluded that other than divalent heavy metal cations, As(3+) appears to be one of the most toxic xenobiotics to platelet function.
Abstract: In vitro effect of mercury (Hg2+), cadmium (Cd2+), and arsenic (As3+) on adenylate cyclase (AC) and phosphodiesterase (PDE) activity in relation to platelet aggregation (PA) was studied in rats. Cd2+ significantly elevated cAMP (p < 0.005) in a dose-dependent (5, 10 and 20 pmoles) manner while Hg2+ and As3+ significantly reduced the cAMP level (p < 0.01 and p < 0.005, respectively). Our studies further reveal that Hg2+ and As3+ inhibit AC and stimulate PDE activity with a concomitant increase in the rate of PA. On the other hand, Cd2+ stimulates AC and inhibits PDE activity with a decrease in the rate of PA. The present investigation suggests that cellular cAMP is a regulatory molecule in the event of PA and the disruption of its homeostasis is directly correlated to xenobiotic effects on PA. It is concluded that other than divalent heavy metal cations, As3+ appears to be one of the most toxic xenobiotics to platelet function.
TL;DR: Results indicate that C-ANP(4-23) treatment of A10 cells desensitizes ANP-C receptor-mediated inhibition of adenylyl cyclase which may be due to the downregulation of ANP -C receptor and decreased expression of Gialpha proteins to which these receptors are coupled.
Abstract: Atrial natriuretic peptide (ANP) receptors A and B are guanylyl cyclase receptors, whereas ANP-C receptors are coupled to adenylyl cyclase through inhibitory guanine nucleotide (Gi) protein. ANP has been shown to downregulate ANP-A and -B receptors and cGMP response in various tissues. In the present studies, we have examined the regulation of ANP-C receptor−adenylyl cyclase signal transduction by ANP and [des(Gln18,Ser19,Gln20,Leu21,Gly22)ANP4-23-NH2](C-ANP4-23) that interacts specifically with ANP-C receptor in A10 smooth muscle cells (SMC). Treatment of the cells with C-ANP4-23 for 24 h resulted in a reduction in ANP receptor binding activity. [125I]ANP99-126 bound to control and C-ANP4-23-treated cell membranes at a single site with dissociation constants of 33.7 ± 6 and 35.0 ± 4.5 pM and Bmax of 74.0 ± 5.0 and 57.6 ± 4.0 fmol/mg of protein, respectively. C-ANP4-23 inhibited adenylyl cyclase activity in a concentration-dependent manner in control cells. A maximal inhibition observed was about 30−40% w...
TL;DR: The results suggest that functional receptors like lysophospholipid receptor Edg-1, which can inhibit adenylate cyclase via Gi protein, are lacking in the rat heart, and will provide a clue to better understand the various types of Sph-1-P-related pathophysiological processes.
TL;DR: The results demonstrated the significant modulations of both receptor binding affinity and transduction (adenylate cyclase activity) that can accompany regional conformational constraints even in larger polypeptide ligands.
Abstract: Glucagon was systematically modified by forming lactam bridges within the central region of the molecule to give conformationally constrained cyclic analogues. Six cyclic glucagon analogues have been designed and synthesized. They are c[Asp(9),Lys(12)][Lys(17,18), Glu(21)]glucagon-NH(2) (1), c[Asp(9),Lys(12)]glucagon-NH(2) (2), c[Lys(12),Asp(15)]glucagon-NH(2) (3), c[Asp(15), Lys(18)]glucagon-NH(2) (4), [Lys(17)-c[Lys(18), Glu(21)]glucagon-NH(2) (5), and c[Lys(12),Asp(21)]glucagon-NH(2) (6). The receptor binding potencies and receptor second messenger activities were determined by radio-receptor binding assays and adenylate cyclase assays, respectively, using rat liver plasma membranes. Most interestingly, analogues 1, 2, 3, and 4 were antagonists of glucagon stimulated adenylate cyclase activity, whereas analogues 5 and 6 were partial agonists in the functional assay. All of the cyclic analogues were found to have reduced binding potencies relative to glucagon. The structural features that might be responsible for these effects were studied using circular dichroism spectroscopy and molecular modeling. These results demonstrated the significant modulations of both receptor binding affinity and transduction (adenylate cyclase activity) that can accompany regional conformational constraints even in larger polypeptide ligands. These studies suggest that the entire molecular conformation, including the flexible middle portion, is important for molecular recognition and transduction at the hepatic glucagon receptor.
TL;DR: It is suggested that axon growth involving high levels of GAP-43 is distinct from the growth stimulation which is rapidly induced by cyclic AMP, which was quickly inducible and reversible, could occur in the presence of transcription inhibitors, and did not entail alterations in branching pattern.
TL;DR: The experiments demonstrated that Y495 and Y612 may be involved in the initiation of the cyclization reaction and Y609 in the stabilization and/or positioning of the intermediate carbocations.
Abstract: The catalytic cavity of Alicyclobacillus acidocaldarius squalene–hopene cyclase is mainly lined by aromatic amino acids. In recombinant cyclases, three out of four tyrosine residues (Y) have been mutated to phenylalanine residues (F). The mutant cyclases Y495F and Y612F had less activity than the wild-type cyclase, but a wild-type product pattern. Mutant Y609F had wild-type activity but a drastically altered product pattern with hopene and significant amounts of bicyclic α-polypodatetraene and different tetracyclic triterpenes (dammaradienes and eupha-7,24-diene). The experiments demonstrated that Y495 and Y612 may be involved in the initiation of the cyclization reaction and Y609 in the stabilization and/or positioning of the intermediate carbocations.
TL;DR: The gene snoaL, encoding a nogalonic acid methyl ester cyclase for nogAlamycin, was used to generate nogsalamycinone, demonstrating that the single cyclase dictates the C-9 stereochemistry of anthracyclines.
Abstract: Nogalamycin is an anthracycline antibiotic produced by Streptomyces nogalater. Its aglycone has a unique stereochemistry (7S, 9S, 10R) compared to that of most other anthracyclines (7S, 9R, 10R). The gene snoaL, encoding a nogalonic acid methyl ester cyclase for nogalamycin, was used to generate nogalamycinone, demonstrating that the single cyclase dictates the C-9 stereochemistry of anthracyclines.
Journal Article•
TL;DR: Isolation of fil-type mutations appears a promising avenue for development of industrial yeast strains with improved stress resistance during active fermentation, and it is shown that high stress resistance and high fermentation activity are compatible biological properties.
Abstract: The initiation of fermentation in the yeast Saccharomyces cerevisiae is associated with a rapid drop in stress resistance. This is disadvantageous for several biotechnological applications, e.g. the preparation of freeze doughs. We have isolated mutants in a laboratory strain which are deficient in fermentation-induced loss of stress resistance (‘fil’ mutants) using a heat shock selection protocol. We show that the fil1 mutant contains a mutation in the CYR1 gene which encodes adenylate cyclase. It causes a change at position 1682 of glutamate into lysine and results in a tenfold drop in adenylate cyclase activity. The fil1 mutant displays a reduction in the glucoseinduced cAMP increase, trehalase activation and loss of heat resistance. Interestingly, the fil1 mutant shows the same growth and fermentation rate as the wild type strain, as opposed to other mutants with reduced activity of the cAMP pathway. Introduction of the fil1 mutation in the vigorous Y55 strain and cultivation of the mutant under pilot scale conditions resulted in a yeast that displayed a higher freeze and drought resistance during active fermentation compared to the wild type Y55 strain. These results show that high stress resistance and high fermentation activity are compatible biological properties. Isolation of fil-type mutations appears a promising avenue for development of industrial yeast strains with improved stress resistance during active fermentation.