Studies on the biosynthesis of cordycepin

The carboxyl carbons of acetate and isovalerate do not serve to any appreciable extent as precursors of cordycepin, thus arguing against the origin of cordycepose from pathways of isoprenoid biosynthesis. Exogenously added [8-14C]adenine, [I-14C]-glucose, and [6-14C]glucose serve as excellent precursors, the latter two compounds labeling both the base and sugar of the nucleoside. [6-14C]glucose labels cordycepose better than [I-14C]glucose, thereby indicating a loss of carbon atom number one of the glucose molecule in cordycepose synthesis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/32376/1/0000451.pd

The inhibition of phosphoribosyl-pyrophosphate amidotransferase activity by cordycepin monophosphate

1. 1. Cordycepin monophosphate has been shown to inhibit the activity of phosphoribosyl-pyrophosphate amidotransferase (ribosylamine-5-phosphate:phyrophosphate phosphoribosyltransferase (glutamate-amidating), EC 2.4.2.14) obtained from both pigeon liver and Bacillus subtilis.2. 2. Evidence is presented for the existence of a phosphorylated derivative of cordycepin in Bacillus subtilis cells which were grown in the presence of cordycepin.3. 3. These results, accompanied by previous evidence, suggest that the inhibition of this enzymatic reaction may be the mechanism by which cordycepin inhibits the growth of Bacillus subtilis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/32099/1/0000149.pd

Isolation and identification of 3'-amino-3'-deoxyadenosine from Cordyceps militaris

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/32269/1/0000331.pd

An improved method of isolation and determination of cordycepin

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/32414/1/0000491.pd

Studies on the synthesis and structure of cordycepin monophosphate

Cordycepin monophosphate has been synthesized by two methods. One of these was chemical and involved treatment of cordycepin with cyanoethyl phosphate in the presence of N,N'-dicyclohexylcarbodiimide with subsequent alkaline hydrolysis. The other involved a phosphate transfer from uridine 5'-phosphate to cordycepin utilizing an enzyme prepared from fresh wheat sprouts. In each instance, the product isolated proved to be the monophosphate derivative and the position of attachment of the phosphate group was shown to be on the primary hydroxyl group of the sugar.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/32257/1/0000319.pd

Studies on the synthesis and structure of cordycepin monophosphate

Cordycepin monophosphate has been synthesized by two methods. One of these was chemical and involved treatment of cordycepin with cyanoethyl phosphate in the presence of N,N'-dicyclohexylcarbodiimide with subsequent alkaline hydrolysis. The other involved a phosphate transfer from uridine 5'-phosphate to cordycepin utilizing an enzyme prepared from fresh wheat sprouts. In each instance, the product isolated proved to be the monophosphate derivative and the position of attachment of the phosphate group was shown to be on the primary hydroxyl group of the sugar.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/32256/1/0000318.pd

Heterologous expression analyses of rice OsCAS in Arabidopsis and in yeast provide evidence for its roles in cyanide detoxification rather than in cysteine synthesis in vivo

While most dicot plants produce little ethylene in their vegetative stage, many monocots such as rice liberate a relatively large amount of ethylene with cyanide as a co-product in their seedling stage when etiolated. One of the known functions of β-cyanoalanine synthase (CAS) is to detoxify the co-product cyanide during ethylene biosynthesis in higher plants. Based on a tryptic peptide sequence obtained from a partially purified CAS activity protein preparation in etiolated rice seedlings, the full-length putative rice CAS-encoding cDNA sequence (OsCAS), which is homologous to those O-acetylserine sulphydrylase (OASS) genes, was cloned. Unlike most of the CAS genes reported from dicots, the transcription of OsCAS is promoted by auxins but suppressed by ethylene. To address the function and the subcellular localization of this gene product in planta, a binary vector construct consisting of this gene appended with a yellow fluorescent protein-encoding sequence was employed to transform Arabidopsis. Specific activities on CAS and OASS of the purified recombinant protein from transgenic Arabidopsis were 181.04 μmol H2S mg−1 protein min−1 and 0.92 μmol Cys mg−1 protein min−1, respectively, indicating that OsCAS favours CAS activity. The subcellular localization of OsCAS was found mostly in the mitochondria by immunogold electron-microscopy. Chemical cross-linking and in-gel assay on a heterodimer composed of functional and non-functional mutants in a yeast expression system on OsCAS suggested that OsCAS functions as a homodimer, similar to that of OASS. Despite the structural similarity of OsCAS with OASS, it has also been confirmed that OsCAS could not interact with serine-acetyltransferase, indicating that OsCAS mainly functions in cyanide detoxification

A Conserved Mechanism for Sulfonucleotide Reduction

Sulfonucleotide reductases are a diverse family of enzymes that catalyze the first committed step of reductive sulfur assimilation. In this reaction, activated sulfate in the context of adenosine-5′-phosphosulfate (APS) or 3′-phosphoadenosine 5′-phosphosulfate (PAPS) is converted to sulfite with reducing equivalents from thioredoxin. The sulfite generated in this reaction is utilized in bacteria and plants for the eventual production of essential biomolecules such as cysteine and coenzyme A. Humans do not possess a homologous metabolic pathway, and thus, these enzymes represent attractive targets for therapeutic intervention. Here we studied the mechanism of sulfonucleotide reduction by APS reductase from the human pathogen Mycobacterium tuberculosis, using a combination of mass spectrometry and biochemical approaches. The results support the hypothesis of a two-step mechanism in which the sulfonucleotide first undergoes rapid nucleophilic attack to form an enzyme-thiosulfonate (E-Cys-S-SO(3) (−)) intermediate. Sulfite is then released in a thioredoxin-dependent manner. Other sulfonucleotide reductases from structurally divergent subclasses appear to use the same mechanism, suggesting that this family of enzymes has evolved from a common ancestor

Integrative inference of gene-regulatory networks in Escherichia coli using information theoretic concepts and sequence analysis

<p>Abstract</p> <p>Background</p> <p>Although <it>Escherichia coli </it>is one of the best studied model organisms, a comprehensive understanding of its gene regulation is not yet achieved. There exist many approaches to reconstruct regulatory interaction networks from gene expression experiments. Mutual information based approaches are most useful for large-scale network inference.</p> <p>Results</p> <p>We used a three-step approach in which we combined gene regulatory network inference based on directed information (DTI) and sequence analysis. DTI values were calculated on a set of gene expression profiles from 19 time course experiments extracted from the Many Microbes Microarray Database. Focusing on influences between pairs of genes in which one partner encodes a transcription factor (TF) we derived a network which contains 878 TF - gene interactions of which 166 are known according to RegulonDB. Afterward, we selected a subset of 109 interactions that could be confirmed by the presence of a phylogenetically conserved binding site of the respective regulator. By this second step, the fraction of known interactions increased from 19% to 60%. In the last step, we checked the 44 of the 109 interactions not yet included in RegulonDB for functional relationships between the regulator and the target and, thus, obtained ten TF - target gene interactions. Five of them concern the regulator LexA and have already been reported in the literature. The remaining five influences describe regulations by Fis (with two novel targets), PhdR, PhoP, and KdgR. For the validation of our approach, one of them, the regulation of lipoate synthase (LipA) by the pyruvate-sensing pyruvate dehydrogenate repressor (PdhR), was experimentally checked and confirmed.</p> <p>Conclusions</p> <p>We predicted a set of five novel TF - target gene interactions in <it>E. coli</it>. One of them, the regulation of <it>lipA </it>by the transcriptional regulator PdhR was validated experimentally. Furthermore, we developed DTInfer, a new R-package for the inference of gene-regulatory networks from microarrays using directed information.</p

Biosynthesis of CdS Quantum Dots Mediated by Volatile Sulfur Compounds Released by Antarctic Pseudomonas fragi

Previously we reported the biosynthesis of intracellular cadmium sulfide quantum dots (CdS QDs) at low temperatures by the Antarctic strain Pseudomonas fragi GC01. Here we studied the role of volatile sulfur compounds (VSCs) in the biosynthesis of CdS QDs by P. fragi GC01. The biosynthesis of nanoparticles was evaluated in the presence of sulfate, sulfite, thiosulfate, sulfide, cysteine and methionine as sole sulfur sources. Intracellular biosynthesis occurred with all sulfur sources tested. However, extracellular biosynthesis was observed only in cultures amended with cysteine (Cys) and methionine (Met). Extracellular nanoparticles were characterized by dynamic light scattering, absorption and emission spectra, energy dispersive X-ray, atomic force microscopy, transmission electron microscopy, X-ray diffraction and X-ray photoelectron spectroscopy. Purified QDs correspond to cubic nanocrystals of CdS with sizes between 2 and 16 nm. The analysis of VSCs revealed that P. fragi GC01 produced hydrogen sulfide (H2S), methanethiol (MeSH) and dimethyl sulfide (DMS) in the presence of sulfate, Met or Cys. Dimethyl disulfide (DMDS) was only detected in the presence of Met. Interestingly, MeSH was the main VSC produced in this condition. In addition, MeSH was the only VSC for which the concentration decreased in the presence of cadmium (Cd) of all the sulfur sources tested, suggesting that this gas interacts with Cd to form nanoparticles. The role of MeSH and DMS on Cds QDs biosynthesis was evaluated in two mutants of the Antarctic strain Pseudomonas deceptionensis M1T: megL- (unable to produce MeSH from Met) and mddA- (unable to generate DMS from MeSH). No biosynthesis of QDs was observed in the megL- strain, confirming the importance of MeSH in QD biosynthesis. In addition, the production of QDs in the mddA- strain was not affected, indicating that DMS is not a substrate for the biosynthesis of nanoparticles. Here, we confirm a link between MeSH production and CdS QDs biosynthesis when Met is used as sole sulfur source. This work represents the first report that directly associates the production of MeSH with the bacterial synthesis of QDs, thus revealing the importance of different VSCs in the biological generation of metal sulfide nanostructures