scholarly journals The role of the pseudo-disaccharide neamine as an intermediate in the biosynthesis of neomycin

1976 ◽  
Vol 159 (3) ◽  
pp. 601-606 ◽  
Author(s):  
C J Pearce ◽  
J E G. Barnett ◽  
C Anthony ◽  
M Akhtar ◽  
S D Gero
Keyword(s):  
Incubation Medium ◽  
Streptomyces Rimosus ◽  
Wild Type ◽  
Streptomyces Fradiae ◽  
Subunit Assembly ◽  
Subsequent Hydrolysis ◽  
Hydrolysis Of

By using wild-type and deoxystreptamine-negative mutants of Streptomyces fradiae grown in media containing [6(-3)H]glucose or [U-14C]glucose, and by subsequent hydrolysis of the labelled neomycin produced, neamines labelled with 3H in both rings I and II, but with 14C in ring I only, were prepared. A mixture of these two forms of neamine was converted by deoxystreptamine-negative Streptomyces rimosus forma paromomycinus into neomycin (not paromomycin) with a 30% yield. The3H: 14C ratio in this neomycin was the same as the measured in neamine produced by hydrolysis of the neomycin, and in unused neamine reisolated from the incubation medium. The 3H:14C ratio in the neomycin was not affected by the presence of unlabelled deoxystreptamine during the incubation. The radioactivity in the neomycin was associated with rings I and II only. It is concluded that the added neamine is incorporated into antibiotic intact, without initial hydrolysis, and that the probable first step in the subunit assembly of neomycin is the formation of neamine.

Genetic characterization of enzymes involved in the priming steps of oxytetracycline biosynthesis in Streptomyces rimosus

Microbiology ◽  
2011 ◽  
Vol 157 (8) ◽  
pp. 2401-2409 ◽  
Author(s):  
Peng Wang ◽  
Xue Gao ◽  
Yit-Heng Chooi ◽  
Zixin Deng ◽  
Yi Tang
Keyword(s):  
Homology Modelling ◽  
Streptomyces Rimosus ◽  
Wild Type ◽  
Aromatic Polyketides ◽  
Starter Unit ◽  
Priming Process ◽  
Hydrolysis Of ◽  
Bacterial Type

Tetracyclines are clinically important aromatic polyketides whose biosynthesis is catalysed by bacterial type II polyketide synthases (PKSs). Tetracyclines are biosynthesized starting with an amide-containing malonamate starter unit and the resulting C-2 carboxyamide is critical for the antibiotic activities. In this work, we genetically verified that an amidotransferase, OxyD, and a thiolase, OxyP, are involved in the biosynthesis and incorporation of the starter unit. First, two mutations, R248T and D268N, were found to be present in OxyD* encoded in Streptomyces rimosus ATCC 13224, a strain that produces the acetate-primed 2-acetyl-2-decarboxyamido-oxytetracycline (ADOTC) instead of the malonamate-primed oxytetracycline (OTC). Homology modelling suggested that in particular D268N may inactivate OxyD. Complementation of S. rimosus ATCC 13224 with wild-type OxyD restored OTC biosynthesis, thereby confirming the essential role of OxyD in the synthesis of the amide starter unit. Second, using a series of knockout and complementation approaches, we demonstrated that OxyP is most likely involved in maintaining fidelity of the amide-priming process via hydrolysis of the competing acetate priming starter units. While the inactivation of OxyP does not eliminate OTC biosynthesis, the ratio of acetate-primed ADOTC to malonamate-primed OTC is significantly increased. This suggests that OxyP plays an ancillary role in OTC biosynthesis and is important for minimizing the levels of ADOTC, a shunt product that has much weaker antibiotic activities than OTC.

scholarly journals Directed Evolution of Clostridium thermocellum β-Glucosidase A Towards Enhanced Thermostability

2019 ◽  
Vol 20 (19) ◽  
pp. 4701 ◽  
Author(s):  
Shahar Yoav ◽  
Johanna Stern ◽  
Orly Salama-Alber ◽  
Felix Frolow ◽  
Michael Anbar ◽  
...  
Keyword(s):  
Clostridium Thermocellum ◽  
Random Mutagenesis ◽  
Cellulose Hydrolysis ◽  
Inhibitory Effect ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Glucose Yield ◽  
Multiple Alignments ◽  
Subsequent Hydrolysis ◽  
Hydrolysis Of

β-Glucosidases are key enzymes in the process of cellulose utilization. It is the last enzyme in the cellulose hydrolysis chain, which converts cellobiose to glucose. Since cellobiose is known to have a feedback inhibitory effect on a variety of cellulases, β-glucosidase can prevent this inhibition by hydrolyzing cellobiose to non-inhibitory glucose. While the optimal temperature of the Clostridium thermocellum cellulosome is 70 °C, C. thermocellum β-glucosidase A is almost inactive at such high temperatures. Thus, in the current study, a random mutagenesis directed evolutionary approach was conducted to produce a thermostable mutant with Kcat and Km, similar to those of the wild-type enzyme. The resultant mutant contained two mutations, A17S and K268N, but only the former was found to affect thermostability, whereby the inflection temperature (Ti) was increased by 6.4 °C. A17 is located near the central cavity of the native enzyme. Interestingly, multiple alignments revealed that position 17 is relatively conserved, whereby alanine is replaced only by serine. Upon the addition of the thermostable mutant to the C. thermocellum secretome for subsequent hydrolysis of microcrystalline cellulose at 70 °C, a higher soluble glucose yield (243%) was obtained compared to the activity of the secretome supplemented with the wild-type enzyme.

scholarly journals In vivo hydrolysis of S-adenosylmethionine induces the met regulon of Escherichia coli

Microbiology ◽  
2006 ◽  
Vol 152 (5) ◽  
pp. 1451-1459 ◽  
Author(s):  
Bernadette L. LaMonte ◽  
Jeffrey A. Hughes
Keyword(s):  
Escherichia Coli ◽  
Plasmid Vector ◽  
Wild Type ◽  
Methionine Biosynthesis ◽  
Induced Expression ◽  
Defined Media ◽  
Sam Synthetase ◽  
Hydrolysis Of

Regulation of methionine biosynthesis in Escherichia coli involves a complex of the MetJ aporepressor protein and S-adenosylmethionine (SAM) repressing expression of most genes in the met regulon. To test the role of SAM in the regulation of met genes directly, SAM pools were depleted by the in vivo expression of the cloned plasmid vector-based coliphage T3 SAM hydrolase (SAMase) gene. Cultures with in vivo SAMase activity were assayed for expression of the metA, B, C, E, F, H, J, K and R genes in cells grown in methionine-rich complete media as well as in defined media with and without l-methionine. In vivo SAMase activity dramatically induced expression between 11- and nearly 1000-fold depending on the gene assayed for all but metJ and metH, and these genes were induced over twofold. metJ : : Tn5 (aporepressor defective) and metK : : Tn5 (SAM synthetase impaired; produces <5 % of wild-type SAM) strains containing in vivo SAMase activity produced even higher met gene activity than that seen in comparably prepared cells with wild-type genes for all but metJ in a MetJ-deficient background. The SAMase-mediated hyperinduction of metH in wild-type cells and of the met genes assayed in metJ : : Tn5 and metK : : Tn5 cells provokes questions about how other elements such as the MetR activator protein or factors beyond the met regulon itself might be involved in the regulation of genes responsible for methionine biosynthesis.

scholarly journals Effect of directional pulling on mechanical protein degradation by ATP-dependent proteolytic machines

2017 ◽  
Vol 114 (31) ◽  
pp. E6306-E6313 ◽  
Author(s):  
Adrian O. Olivares ◽  
Hema Chandra Kotamarthi ◽  
Benjamin J. Stein ◽  
Robert T. Sauer ◽  
Tania A. Baker
Keyword(s):  
Escherichia Coli ◽  
Protein Degradation ◽  
Single Molecule ◽  
Wild Type ◽  
N Terminus ◽  
Multiple Copies ◽  
Rate Limiting ◽  
Hydrolysis Of ◽  
Translocation Speed

AAA+ proteases and remodeling machines couple hydrolysis of ATP to mechanical unfolding and translocation of proteins following recognition of sequence tags called degrons. Here, we use single-molecule optical trapping to determine the mechanochemistry of two AAA+ proteases, Escherichia coli ClpXP and ClpAP, as they unfold and translocate substrates containing multiple copies of the titinI27 domain during degradation initiated from the N terminus. Previous studies characterized degradation of related substrates with C-terminal degrons. We find that ClpXP and ClpAP unfold the wild-type titinI27 domain and a destabilized variant far more rapidly when pulling from the N terminus, whereas translocation speed is reduced only modestly in the N-to-C direction. These measurements establish the role of directionality in mechanical protein degradation, show that degron placement can change whether unfolding or translocation is rate limiting, and establish that one or a few power strokes are sufficient to unfold some protein domains.

scholarly journals Essential role of endogenously synthesized tylosin for induction of ermSF in Streptomyces fradiae.

1997 ◽  
Vol 41 (5) ◽  
pp. 1203-1205 ◽  
Author(s):  
E Memili ◽  
B Weisblum
Keyword(s):  
Essential Role ◽  
Wild Type ◽  
Streptomyces Fradiae

We compared ermSF induction in wild-type Streptomyces fradiae NRRL B-2702 and that in GS-14, a tylA mutant which cannot synthesize tylosin. Our findings suggest that (i) endogenously synthesized tylosin plays an obligatory role in ermSF induction and (ii) tylosin, or a biosynthetic intermediate beyond tylactone, has an "autocrine" function that induces ErmSF synthesis, thereby enabling S. fradiae to resist higher levels of tylosin.

Differential Role of Components of the Fibrinolytic System in the Formation and Removal of Thrombus Induced by Endothelial Injury

1999 ◽  
Vol 81 (04) ◽  
pp. 601-604 ◽  
Author(s):  
Hiroyuki Matsuno ◽  
Osamu Kozawa ◽  
Masayuki Niwa ◽  
Shigeru Ueshima ◽  
Osamu Matsuo ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Thrombus Formation ◽  
Endothelial Injury ◽  
Fibrinolytic System ◽  
Tissue Type ◽  
Clot Lysis ◽  
Wild Type ◽  
Type Plasminogen Activator ◽  
Pai 1

SummaryThe role of fibrinolytic system components in thrombus formation and removal in vivo was investigated in groups of six mice deficient in urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA), or plasminogen activator inhibitor-1 (PAI-1) (u-PA-/-, t-PA-/- or PAI-1-/-, respectively) or of their wild type controls (u-PA+/+, t-PA+/+ or PAI-1+/+). Thrombus was induced in the murine carotid artery by endothelial injury using the photochemical reaction between rose bengal and green light (540 nm). Blood flow was continuously monitored for 90 min on day 0 and for 20 min on days 1, 2 and 3. The times to occlusion after the initiation of endothelial injury in u-PA+/+, t-PA+/+ or PAI-1+/+ mice were 9.4 ± 1.3, 9.8 ± 1.1 or 9.7 ± 1.6 min, respectively. u-PA-/- and t-PA-/- mice were indistinguishable from controls, whereas that of PAI-1-/- mice were significantly prolonged (18.4 ± 3.7 min). Occlusion persisted for the initial 90 min observation period in 10 of 18 wild type mice and was followed by cyclic reflow and reocclusion in the remaining 8 mice. At day 1, persistent occlusion was observed in 1 wild type mouse, 8 mice had cyclic reflow and reocclusion and 9 mice had persistent reflow. At day 2, all injured arteries had persistent reflow. Persistent occlusion for 90 min on day 0 was observed in 3 u-PA-/-, in all t-PA-/- mice at day 1 and in 2 of the t-PA-/-mice at day 2 (p <0.01 versus wild type mice). Persistent patency was observed in all PAI-1-/- mice at day 1 and in 5 of the 6 u-PA-/- mice at day 2 (both p <0.05 versus wild type mice). In conclusion, t-PA increases the rate of clot lysis after endothelial injury, PAI-1 reduces the time to occlusion and delays clot lysis, whereas u-PA has little effect on thrombus formation and spontaneous lysis.

scholarly journals Salicylic Acid Accumulation Controlled by LSD1 Is Essential in Triggering Cell Death in Response to Abiotic Stress

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 962
Author(s):  
Maciej Jerzy Bernacki ◽  
Anna Rusaczonek ◽  
Weronika Czarnocka ◽  
Stanisław Karpiński
Keyword(s):  
Cell Death ◽  
Salicylic Acid ◽  
Abiotic Stress ◽  
Wild Type ◽  
Pr Genes ◽  
Genes Expression ◽  
Salicylic Acid Accumulation ◽  
Cell Death Phenotype ◽  
Insight Into

Salicylic acid (SA) is well known hormonal molecule involved in cell death regulation. In response to a broad range of environmental factors (e.g., high light, UV, pathogens attack), plants accumulate SA, which participates in cell death induction and spread in some foliar cells. LESION SIMULATING DISEASE 1 (LSD1) is one of the best-known cell death regulators in Arabidopsis thaliana. The lsd1 mutant, lacking functional LSD1 protein, accumulates SA and is conditionally susceptible to many biotic and abiotic stresses. In order to get more insight into the role of LSD1-dependent regulation of SA accumulation during cell death, we crossed the lsd1 with the sid2 mutant, caring mutation in ISOCHORISMATE SYNTHASE 1(ICS1) gene and having deregulated SA synthesis, and with plants expressing the bacterial nahG gene and thus decomposing SA to catechol. In response to UV A+B irradiation, the lsd1 mutant exhibited clear cell death phenotype, which was reversed in lsd1/sid2 and lsd1/NahG plants. The expression of PR-genes and the H2O2 content in UV-treated lsd1 were significantly higher when compared with the wild type. In contrast, lsd1/sid2 and lsd1/NahG plants demonstrated comparability with the wild-type level of PR-genes expression and H2O2. Our results demonstrate that SA accumulation is crucial for triggering cell death in lsd1, while the reduction of excessive SA accumulation may lead to a greater tolerance toward abiotic stress.

scholarly journals The Prognostic and Predictive Role of Somatic BRCA Mutations in Ovarian Cancer: Results from a Multicenter Cohort Study

Diagnostics ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 565
Author(s):  
Angela Toss ◽  
Claudia Piombino ◽  
Elena Tenedini ◽  
Alessandra Bologna ◽  
Elisa Gasparini ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Positive Impact ◽  
Progression Free Survival ◽  
Molecular Testing ◽  
Wild Type ◽  
Brca Mutations ◽  
Multicenter Cohort Study ◽  
Advanced Stages ◽  
Predictive Role

Previous research involving epithelial ovarian cancer patients showed that, compared to germline BRCA (gBRCA) mutations, somatic BRCA (sBRCA) mutations present a similar positive impact with regard to overall survival (OS) and platinum and PARP (poly (ADP-ribose) polymerase) inhibitor sensitivity. Nevertheless, molecular testing in these studies did not include copy number variation (CNV) analyses of BRCA genes. The aim of this study was to explore the prognostic and predictive role of sBRCA mutations as compared to gBRCA mutations in patients who were also tested for CNVs. Among the 158 patients included in the study, 17.09% of patients carried a pathogenic or likely pathogenic gBRCA variant and 15.19% of patients presented pathogenetic or likely pathogenic sBRCA variants and/or CNVs. Overall, 81.6% of the patients included in this study were diagnosed with a serous histotype, and 77.2% were in advanced stages. Among women diagnosed in advanced stages, gBRCA patients showed better progression-free survival and OS as compared to sBRCA and wild-type patients, whereas sBRCA patients did not show any advantage in outcome as compared to wild-type patients. In this study, the introduction of CNV analyses increased the detection rate of sBRCA mutations, and the resulting classification among gBRCA, sBRCA and wild-type patients was able to properly stratify the prognosis of OC patients. Particularly, sBRCA mutation patients failed to show any outcome advantage as compared to wild-type patients.

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