scholarly journals Mechanism of 4-(β-D-Ribofuranosyl)aminobenzene 5′-Phosphate Synthase, a Key Enzyme in the Methanopterin Biosynthetic Pathway

2004 ◽  
Vol 279 (38) ◽  
pp. 39389-39395 ◽  
Author(s):  
Razvan V. Dumitru ◽  
Stephen W. Ragsdale
Keyword(s):  
Biosynthetic Pathway ◽  
Key Enzyme ◽  
Phosphate Synthase

Disruption of genes for the enhanced biosynthesis of α-ketoglutarate in Corynebacterium glutamicum

2012 ◽  
Vol 58 (3) ◽  
pp. 278-286 ◽  
Author(s):  
Jae-Hyung Jo ◽  
Hye-Young Seol ◽  
Yun-Bom Lee ◽  
Min-Hong Kim ◽  
Hyung-Hwan Hyun ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Biosynthetic Pathway ◽  
Isocitrate Lyase ◽  
Glutamate Synthase ◽  
Flask Culture ◽  
Control Strain ◽  
Enhanced Production ◽  
Key Enzyme ◽  
Microbial Strains ◽  
Glyoxylate Pathway

The development of microbial strains for the enhanced production of α-ketoglutarate (α-KG) was investigated using a strain of Corynebacterium glutamicum that overproduces of l-glutamate, by disrupting three genes involved in the α-KG biosynthetic pathway. The pathways competing with the biosynthesis of α-KG were blocked by knocking out aceA (encoding isocitrate lyase, ICL), gdh (encoding glutamate dehydrogenase, l-gluDH), and gltB (encoding glutamate synthase or glutamate-2-oxoglutarate aminotransferase, GOGAT). The strain with aceA, gltB, and gdh disrupted showed reduced ICL activity and no GOGAT and l-gluDH activities, resulting in up to 16-fold more α-KG production than the control strain in flask culture. These results suggest that l-gluDH is the key enzyme in the conversion of α-KG to l-glutamate; therefore, prevention of this step could promote α-KG accumulation. The inactivation of ICL leads the carbon flow to α-KG by blocking the glyoxylate pathway. However, the disruption of gltB did not affect the biosynthesis of α-KG. Our results can be applied in the industrial production of α-KG by using C. glutamicum as producer.

scholarly journals The biosynthetic pathway of potato solanidanes diverged from that of spirosolanes due to evolution of a dioxygenase

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ryota Akiyama ◽  
Bunta Watanabe ◽  
Masaru Nakayasu ◽  
Hyoung Jae Lee ◽  
Junpei Kato ◽  
...  
Keyword(s):  
Solanum Tuberosum ◽  
Solanum Lycopersicum ◽  
Biosynthetic Pathway ◽  
Potato Breeding ◽  
Chemical Diversity ◽  
Evolutionary Divergence ◽  
Food Crop ◽  
Common Pathway ◽  
Structural Differences ◽  
Key Enzyme

AbstractPotato (Solanum tuberosum), a worldwide major food crop, produces the toxic, bitter tasting solanidane glycoalkaloids α-solanine and α-chaconine. Controlling levels of glycoalkaloids is an important focus on potato breeding. Tomato (Solanum lycopersicum) contains a bitter spirosolane glycoalkaloid, α-tomatine. These glycoalkaloids are biosynthesized from cholesterol via a partly common pathway, although the mechanisms giving rise to the structural differences between solanidane and spirosolane remained elusive. Here we identify a 2-oxoglutarate dependent dioxygenase, designated as DPS (Dioxygenase for Potato Solanidane synthesis), that is a key enzyme for solanidane glycoalkaloid biosynthesis in potato. DPS catalyzes the ring-rearrangement from spirosolane to solanidane via C-16 hydroxylation. Evolutionary divergence of spirosolane-metabolizing dioxygenases contributes to the emergence of toxic solanidane glycoalkaloids in potato and the chemical diversity in Solanaceae.

scholarly journals Bornyl Diphosphate Synthase From Cinnamomum burmanni and Its Application for (+)-Borneol Biosynthesis in Yeast

2021 ◽  
Vol 9 ◽  
Author(s):  
Rui Ma ◽  
Ping Su ◽  
Juan Guo ◽  
Baolong Jin ◽  
Qing Ma ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Biosynthetic Pathway ◽  
Main Product ◽  
Microbial Fermentation ◽  
Biosynthesis Pathway ◽  
Analgesic Effects ◽  
Pathway Reconstruction ◽  
Key Enzyme ◽  
Shake Flasks

(+)-Borneol is a desirable monoterpenoid with effective anti-inflammatory and analgesic effects that is known as soft gold. (+)-bornyl diphosphate synthase is the key enzyme in the (+)-borneol biosynthesis pathway. Despite several reported (+)-bornyl diphosphate synthase genes, relatively low (+)-borneol production hinders the attempts to synthesize it using microbial fermentation. Here, we identified the highly specific (+)-bornyl diphosphate synthase CbTPS1 from Cinnamomum burmanni. An in vitro assay showed that (+)-borneol was the main product of CbTPS1 (88.70% of the total products), and the Km value was 5.11 ± 1.70 μM with a kcat value of 0.01 s–1. Further, we reconstituted the (+)-borneol biosynthetic pathway in Saccharomyces cerevisiae. After tailored truncation and adding Kozak sequences, the (+)-borneol yield was improved by 96.33-fold to 2.89 mg⋅L–1 compared with the initial strain in shake flasks. This work is the first reported attempt to produce (+)-borneol by microbial fermentation. It lays a foundation for further pathway reconstruction and metabolic engineering production of this valuable natural monoterpenoid.

scholarly journals Crosstalk among Indoleamines, Neuropeptides and JH/20E in Regulation of Reproduction in the American Cockroach, Periplaneta americana

Insects ◽  
2020 ◽  
Vol 11 (3) ◽  
pp. 155 ◽  
Author(s):  
A. S. M. Kamruzzaman ◽  
Azam Mikani ◽  
Amr A. Mohamed ◽  
Azza M. Elgendy ◽  
Makio Takeda
Keyword(s):  
Oocyte Maturation ◽  
Biosynthetic Pathway ◽  
Periplaneta Americana ◽  
American Cockroach ◽  
Second Step ◽  
Double Stranded Rna ◽  
Vitellogenin Receptor ◽  
Genes Encoding ◽  
Key Enzyme ◽  
Indoleamine Metabolism

Although the regulation of vitellogenesis in insects has been mainly discussed in terms of ‘classical’ lipid hormones, juvenile hormone (JH), and 20-hydroxyecdysone (20E), recent data support the notion that this process must be adjusted in harmony with a nutritional input/reservoir and involvement of certain indoleamines and neuropeptides in regulation of such process. This study focuses on crosstalks among these axes, lipid hormones, monoamines, and neuropeptides in regulation of vitellogenesis in the American cockroach Periplaneta americana with novel aspects in the roles of arylalkylamine N-acetyltransferase (aaNAT), a key enzyme in indoleamine metabolism, and the enteroendocrine peptides; crustacean cardioactive peptide (CCAP) and short neuropeptide F (sNPF). Double-stranded RNA against aaNAT (dsRNAaaNAT) was injected into designated-aged females and the effects were monitored including the expressions of aaNAT itself, vitellogenin 1 and 2 (Vg1 and Vg2) and the vitellogenin receptor (VgR) mRNAs, oocyte maturation and changes in the hemolymph peptide concentrations. Effects of peptides application and 20E were also investigated. Injection of dsRNAaaNAT strongly suppressed oocyte maturation, transcription of Vg1, Vg2, VgR, and genes encoding JH acid- and farnesoate O-methyltransferases (JHAMT and FAMeT, respectively) acting in the JH biosynthetic pathway. However, it did not affect hemolymph concentrations of CCAP and sNPF. Injection of CCAP stimulated, while sNPF suppressed oocyte maturation and Vgs/VgR transcription, i.e., acting as allatomedins. Injection of CCAP promoted, while sNPF repressed ecdysteroid (20E) synthesis, particularly at the second step of Vg uptake. 20E also affected the JH biosynthetic pathway and Vg/VgR synthesis. The results revealed that on the course of vitellogenesis, JH- and 20E-mediated regulation occurs downstream to indoleamines- and peptides-mediated regulations. Intricate mutual interactions of these regulatory routes must orchestrate reproduction in this species at the highest potency.

scholarly journals High-Throughput Screen for Inhibitors of 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase by Surrogate Ligand Competition

2003 ◽  
Vol 8 (3) ◽  
pp. 332-339 ◽  
Author(s):  
Elizabeth B. Gottlin ◽  
R. Edward Benson ◽  
Scott Conary ◽  
Brett Antonio ◽  
Kellie Duke ◽  
...  
Keyword(s):  
High Throughput ◽  
Binding Sites ◽  
Biosynthetic Pathway ◽  
Enzyme Assay ◽  
High Throughput Screen ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Biochemical Assays ◽  
Key Enzyme ◽  
Nadph Oxidation

1-Deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr) is a key enzyme in a biosynthetic pathway for isoprenoids that is unique to eubacteria and plants. Dxr catalyzes the rearrangement and NADPH-dependent reduction of 1-deoxy-D-xylulose 5-phosphate to 2-C-methyl-D-erythritol 4-phosphate. The authors have purified Escherichia coli Dxr and devised a high-throughput screen (HTS) for compounds that bind to this enzyme at a functional site. Evidence is presented that the surrogate ligand directly binds or allosterically affects both the D-1-deoxyxylulose 5-phosphate (DXP) and NADPH binding sites. Compounds that bind at either or both sites that compete for binding with the surrogate ligand register as hits. The time-resolved fluorescence-based assay represents an improvement over the Dxr enzyme assay that relies on relatively insensitive measurements of NADPH oxidation. Screening 32,000 compounds from a diverse historical library, the authors obtained 89 potent inhibitors in the surrogate ligand competition assay. The results presented here suggest that peptide surrogate ligands may be useful in formatting HTS for proteins with difficult biochemical assays or targets of unknown function. ( Journal of Biomolecular Screening 2003:332-339)

scholarly journals Subcellular localization and tissue distribution of sialic acid-forming enzymes. N-acetylneuraminate-9-phosphate synthase and N-acetylneuraminate 9-phosphatase

1984 ◽  
Vol 223 (2) ◽  
pp. 323-328 ◽  
Author(s):  
J Van Rinsum ◽  
W Van Dijk ◽  
G J Hooghwinkel ◽  
W Ferwerda
Keyword(s):  
Subcellular Localization ◽  
Biosynthetic Pathway ◽  
Cytosolic Fraction ◽  
Phosphate Esters ◽  
Rat Tissues ◽  
Acetylneuraminic Acid ◽  
Nitrophenyl Phosphate ◽  
Sucrose Medium ◽  
Hydrolysis Of ◽  
Phosphate Synthase

The activities of N-acetylneuraminate 9-phosphate synthase and N-acetylneuraminate 9-phosphatase, the two enzymes involved in the final steps of the biosynthetic pathway of N-acetylneuraminic acid, were measured with the substrates N-acetyl[14C]mannosamine 6-phosphate and N-acetyl[14C]neuraminic acid 9-phosphate respectively. Subcellular localization studies in rat liver indicated that both enzymes are localized in the cytosolic fraction after homogenization in sucrose medium. To test the possibility of misinterpretation due to the hydrolysis of N-acetylneuraminic acid 9-phosphate by non-specific phosphatases, the hydrolysis of various phosphate esters by the cytosolic fraction was tested. Only p-nitrophenyl phosphate was hydrolysed; however, competition studies with N-acetylneuraminic acid 9-phosphate and p-nitrophenyl phosphate indicated that two different enzymes were involved and that no competition existed between the two substrates. In various other rat tissues N-acetylneuraminate-9-phosphate synthase and N-acetylneuraminate 9-phosphatase activities were detected, suggesting that N-acetylmannosamine 6-phosphate is a general precursor for N-acetylneuraminic acid biosynthesis in all the tissues studied.

scholarly journals TRANSFORMASI GENETIK DAN EKSPRESI MUTAN SUCROSE PHOSPHATE SYNTHASE PADA TANAMAN TOMAT

2019 ◽  
Vol 6 (1) ◽  
pp. 130
Author(s):  
Suwinda Fibriani ◽  
Inyana Dwi Agustien ◽  
Widhi Dyah Sawitri ◽  
Bambang Sugiharto
Keyword(s):  
Genetic Transformation ◽  
Tomato Plant ◽  
Sucrose Phosphate Synthase ◽  
Transgenic Tomato ◽  
Pcr Analysis ◽  
N Terminus ◽  
Key Enzyme ◽  
Sucrose Biosynthesis ◽  
Sucrose Phosphate ◽  
Phosphate Synthase

Genetic Transformation and Expression of Sucrose Phosphate Synthase Mutant in Tomato Plant ABSTRACTSucrose phosphate synthase (SPS) is a key enzyme responsible for sucrose biosynthesis. In its regulation, SPS activity is modulated by an allosteric effector glucose-6-phosphate (G6P) suggested to have an ability to bind SPS N-terminus domain. To understand the role of N-terminus in regulating SPS, the SPS gene was mutated with the deletion of N-terminus domain (∆N-SPS). The ∆N-SPS gen was transformed into tomato plants with 5% transformation efficiency. Three transgenic tomato plant 4.20, 5.5.1, and 5.10 were obtained and confirmed by PCR analysis. Transgenic tomato expression was characterized by enzymatic analysis. Result showed that the G6P allosteric regulation in transgenic ∆N-SPS had lost and the SPS activity increased by 2-fold compared to non-transgenic plant. This showed that N-terminus domain-deleted SPS could be actively expressed in plant. Keywords: enzyme, genetic transformation, N-terminus domain deletion, sucrose phosphate synthase, tomato ABSTRAKSucrose phosphate synthase (SPS) merupakan enzim kunci yang bertanggung jawab dalam sintesis sukrosa. Dalam regulasinya, aktifitas SPS dipengaruhi oleh alosterik efektor glukosa-6-fosfat (G6P) yang diduga dapat berikatan pada domain N-terminus SPS. Untuk mengetahui peran N-terminus pada regulasi SPS, dilakukan mutasi SPS dengan penghilangan domain N-terminus (∆N-SPS). Gen ∆N-SPS diinsersi pada tanaman tomat melalui transformasi genetik dengan efisiensi transformasi 5%. Tiga tanaman transgenik tomat (event4.20; 5.5.1; dan 5.10) didapatkan dan positif terkonfirmasi melalui analisis PCR. Ekspresi mutan dikarakterisasi melalui analisis enzimatik. Hasil menunjukkan bahwa tanaman tomat transgenik ∆N-SPS tidak dipengaruhi regulasi alosterik G6P dan aktifitas SPS 2 kali lipat lebih tinggi daripada tanaman bukan transgenik. Ini menunjukkan bahwa SPS dengan delesi domain N-terminus dapat terekspresi aktif pada tanaman.  Kata Kunci: delesi domain N-terminus, enzim, sucrose phosphate synthase, tomat, transformasi genetik 

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