A high performance distillation system to remove krypton from xenon was constructed, and a purity level of Kr/Xe
=
∼3
×
10
−12 was achieved. This development is crucial in facilitating ...high-sensitivity low-background experiments such as the search for dark matter in the universe.
The Colletotrichum lagenarium PKS1 gene encoding iterative type I polyketide synthase of 1,3,6,8-tetrahydroxynaphthalene (T4HN) was overexpressed in Aspergillus oryzae. SDS−PAGE analysis of the ...cell-free extract prepared from the transformant showed an intense band of 230000 which corresponded to the molecular weight of the deduced PKS1 protein. By using this cell-free extract, in vitro synthesis of T4HN was successfully confirmed as the first example of the fungal multi-aromatic ring polyketide synthase activity ever detected. To identify the starter unit for T4HN synthesis, 14C-labeled acetyl CoA and/or 14C-labeled malonyl CoA were used as substrates for T4HN synthase reaction. Observed was the incorporation of 14C label into T4HN solely from malonyl CoA even in the absence of acetyl CoA and not from acetyl CoA. This in vitro result unambiguously identified that malonyl CoA serves as the starter as well as extender units in the formation of T4HN by fungal polyketide synthase PKS1.
Chalcone synthase (CHS) and stilbene synthase (STS) catalyse condensation reactions of p-coumaroyl-CoA and three C(2) units from malonyl-CoA up to a common tetraketide intermediate but then catalyse ...different cyclization reactions to produce naringenin chalcone and resveratrol respectively. On the basis of sequence alignment with other condensing enzymes including 3-ketoacyl-(acyl carrier protein) synthases of polyketide and fatty-acid synthases, site-directed mutagenesis was performed on the active-site G(372)FGPG loops in CHS and STS. The CHS-P375G mutant showed a 6-fold decrease in overall condensing activity with selectively increased production of p-coumaroyltriacetic acid lactone (CTAL, the derailment product of the tetraketide intermediate). Meanwhile, resveratrol production by STS-P(375)G strongly decreased to give various products in the order CTAL> resveratrol approximately bisnoryangonin>naringenin. As a result, naringenin production (cross-reaction) by STS-P(375)G was close to 30% of resveratrol production. Both G(374)L mutants of CHS and STS showed no condensing activity with residual malonyl-CoA decarboxylase activity. These results suggested that the G(372)FGPG loop in CHS and STS contribute to a determination of the outcome during cyclization reactions by serving as a part of the active-site scaffold on which the stereochemistry of cyclization is performed. These observations provide the first biochemical indication that cyclization reactions are modulated by active-site geometry. The implications for the evolutionary relationship of these enzymes are also discussed.
► A prototype single phase liquid xenon detector was developed and operated. ► We have tested performance of this prototype detector. ► We have measured the physical properties of liquid xenon. ► We ...have established vertex and energy reconstruction methods. ► We have demonstrated liquid xenon’s self-shielding power against γ-rays.
Liquid xenon is a suitable material for a dark matter search. For future large scale experiments, single phase detectors are attractive due to their simple configuration and scalability. However, in order to reduce backgrounds, they need to fully rely on liquid xenon’s self-shielding property. A prototype detector was developed at Kamioka Observatory to establish vertex and energy reconstruction methods and to demonstrate the self-shielding power against γ-rays from outside of the detector. 662keV γ-rays from 137Cs are attenuated by a factor of about 50 over a distance of 20cm.
A class of Streptomyces aromatic polyketide antibiotics, the benzoisochromanequinones, all shows trans stereochemistry at C-3 and C-15 in the pyran ring. The opposite stereochemical control found in ...actinorhodin (3S, 15R, ACT) from S. coelicolor A3(2) and dihydrogranaticin (3R, 15S, DHGRA) from S. violaceoruber Tü22 was studied by functional expression of the potentially relevant ketoreductase genes, actIII, actVI-ORF1, gra-ORF5, and gra-ORF6. A common bicyclic intermediate was postulated to undergo stereospecific reduction to provide either the 3-(S) or the 3-(R) configuration of an advanced intermediate, 4-dihydro-9-hydroxy-1-methyl-10-oxo-3-H-naphtho2,3-cpyran-3-acetic acid (DNPA). Combinations of the four ketoreductase genes were coexpressed with the early biosynthetic genes encoding a type II minimal polyketide synthase, aromatase, and cyclase. gra-ORF6 was essential to produce (R)-DNPA in DHGRA biosynthesis. Out of the various recombinants carrying the relevant ketoreductases, the set of gra-ORF5 and -ORF6 under translational coupling (on pIK191) led to the most efficient production of (R)-DNPA as a single product, implying a possible unique cooperative function whereby gra-ORF6 might encode a "guiding" protein to control the regio- and stereochemical course of reduction at C-3 catalyzed by the gra-ORF5 protein. Updated BLAST-based database analysis suggested that the gra-ORF6 product, a putative short-chain dehydrogenase, has virtually no sequence homology with the actVI-ORF1 protein, which was previously shown to determine the 3-(S) configuration of DNPA in ACT biosynthesis. This demonstrates an example of opposite stereochemical control in antibiotic biosynthesis, providing a key branch point to afford diverse chiral metabolic pools.
Triterpenes exhibit a wide range of structural diversity produced by a sequence of biosynthetic reactions. Cyclization of oxidosqualene is the initial origin of structural diversity of skeletons in ...their biosynthesis, and subsequent regio‐ and stereospecific hydroxylation of the triterpene skeleton produces further structural diversity. The enzymes responsible for this hydroxylation were thought to be cytochrome P450‐dependent monooxygenase, although their cloning has not been reported. To mine these hydroxylases from cytochrome P450 genes, five genes (CYP71D8, CYP82A2, CYP82A3, CYP82A4 and CYP93E1) reported to be elicitor‐inducible genes in Glycine max expressed sequence tags (EST), were amplified by PCR, and screened for their ability to hydroxylate triterpenes (β‐amyrin or sophoradiol) by heterologous expression in the yeast Saccharomyces cerevisiae. Among them, CYP93E1 transformant showed hydroxylating activity on both substrates. The products were identified as olean‐12‐ene‐3β,24‐diol and soyasapogenol B, respectively, by GC‐MS. Co‐expression of CYP93E1 and β‐amyrin synthase in S. cerevisiae yielded olean‐12‐ene‐3β,24‐diol. This is the first identification of triterpene hydroxylase cDNA from any plant species. Successful identification of a β‐amyrin and sophoradiol 24‐hydroxylase from the inducible family of cytochrome P450 genes suggests that other triterpene hydroxylases belong to this family. In addition, substrate specificity with the obtained P450 hydroxylase indicates the two possible biosynthetic routes from triterpene‐monool to triterpene‐triol.
HIV inhibitor from Thai bitter gourd Jiratchariyakul, W; Wiwat, C; Vongsakul, M ...
Planta medica,
06/2001, Letnik:
67, Številka:
4
Journal Article
Recenzirano
Thai bitter gourd protein (MRK29) was isolated from Momordica charantia ripe fruit and seed. The purification was performed by ammonium sulfate fractionation and gel filtration chromatography. MRK29 ...possessed one isoelectric point of (pI) > or = 9, and the time of flight mass spectrum (TOFMS) indicated its molecular weight at 28.6 kD. The twenty amino acid sequence from the N-terminus was in the following order: 1Asp Val Asn Phe Arg Leu Ser Gly Ala 10Asp Pro Arg X Tyr Gly Met Phe Ile Glu 20Asp. MRK29 inhibited the HIV-1 reverse transcriptase with 50% IR at the concentration of 18 micrograms/ml. MRK29 was concentrated in the 30-60% salt precipitated fraction, at which the concentration of 0.175 microgram/ml exerted 82% reduction of viral core protein p24 expression in HIV-infected cells. MRK29 might have modulatory role on immune cells, because it increased 3-fold TNF activity.
Three oxidosqualene cyclase (OSC) cDNAs (
CPX,
CPQ,
CPR) were cloned from seedlings of
Cucurbita pepo by homology based PCR method. Their open reading frames were expressed in lanosterol synthase ...deficient (
erg7)
Saccharomyces cerevisiae strain GIL77. Analyses of in vitro enzyme activities and in vivo accumulated products in the transformants demonstrated that CPQ and CPX encode cucurbitadienol and cycloartenol synthases, respectively. These results indicated the presence of distinct OSCs for cycloartenol and cucurbitadienol synthesis in this plant.
Graphic
Ferns are the most primitive vascular plants. The phytosterols of ferns are the same as those of higher plants, but they produce characteristic triterpenes. The most distinct feature is the lack of ...oxygen functionality at C-3, suggesting that the triterpenes of ferns may be biosynthesized by direct cyclization of squalene. To obtain some insights into the molecular bases for the biosynthesis of triterpenes in ferns, we cloned ACX, an oxidosqualene cyclase homologue, encoding a cycloartenol synthase (CAS) and ACH, a squalene cyclase homologue, encoding a 22-hydroxyhopane synthase from
Adiantum capillus-
veneris. Phylogenetic analysis revealed that ACH is located in the cluster of bacterial SCs, while ACX is in the cluster of higher plant CASs.
Chalcone synthases, which biosynthesize chalcones (the starting materials for many flavonoids,), have been believed to be specific to plants. However, the rppA gene from the Gram-positive, ...soil-living filamentous bacterium Streptomyces griseus encodes a 372-amino-acid protein that shows significant similarity to chalcone synthases. Several rppA-like genes are known, but their functions and catalytic properties have not been described. Here we show that a homodimer of RppA catalyses polyketide synthesis: it selects malonyl-coenzyme-A as the starter, carries out four successive extensions and releases the resulting pentaketide to cyclize to 1,3,6,8-tetrahydroxynaphthalene (THN). Site-directed mutagenesis revealed that, as in other chalcone synthases,, a cysteine residue is essential for enzyme activity. Disruption of the chromosomal rppA gene in S. griseus abolished melanin production in hyphae, resulting in 'albino' mycelium. THN was readily oxidized to form 2,5,7-trihydroxy-1,4-naphthoquinone (flaviolin), which then randomly polymerized to form various coloured compounds. THN formed by RppA appears to be an intermediate in the biosynthetic pathways for not only melanins but also various secondary metabolites containing a naphthoquinone ring. Therefore, RppA is a chalcone-synthase-related synthase that synthesizes polyketides and is found in the Streptomyces and other bacteria.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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