Cholesterol biotransformation to androsta-1,4-diene-3,17-dione by growing cells of <Emphasis Type="Italic">Chryseobacterium gleum</Emphasis>

Cholesterol oxidase activity was studied during biotransformation of cholesterol to androsta-1,4-diene-3,17-dione (ADD) by Chryseobacterium gleum. Spent LB media, containing cholesterol (3 mM≈1 g l⁻¹) where the bacterium was grown for 24 h, at 30°C with constant shaking at 120 rpm, had the highest enzyme activity (167 U mg⁻¹). The growing cells produced 0.076 g ADD from 1 g cholesterol l⁻¹.     

Endophytic fungal strains of Fusarium solani, from Apodytes dimidiata E. Mey. ex Arn (Icacinaceae) produce camptothecin, 10-hydroxycamptothecin and 9-methoxycamptothecin

Camptothecin and 10-hydroxycamptothecin are two important precursors for the synthesis of the clinically useful anticancer drugs, topotecan and irinotecan. In recent years, efforts have been made to identify novel plant and endophytic fungal sources of camptothecin and 10-hydroxycamptothecin. In this study we have isolated endophytic fungi strains from Apodytes dimidiata (Icacinaceae), a medium sized tree from the Western Ghats, India. The fungi were identified as Fusarium solani using both ITS rDNA sequencing and spore morphology. Two strains, MTCC 9667 and MTCC 9668 were isolated, both of which produced camptothecin and 9-methoxycamptothecin in their mycelia; one of the strains, MTCC 9668 also produced 10-hydroxycamptothecin, though in small amounts. The yields of camptothecin in MTCC 9667 and MTCC 9668 were 37 and 53 μg/100 g, respectively, after 4 days of incubation in broth culture. The yields of 10-hydroxycamptothecin and 9-methoxycamptothecin in MTCC 9668 were 8.2 and 44.9 μg/100 g, respectively. Further research in optimizing the culture conditions of these fungal strains might permit their application for the production of camptothecin and 10-hydroxycamptothecin.     

An optimization framework for identifying reaction activation/inhibition or elimination candidates for overproduction in microbial systems

We introduce a computational framework termed OptReg that determines the optimal reaction activations/inhibitions and eliminations for targeted biochemical production. A reaction is deemed up- or downregulated if it is constrained to assume flux values significantly above or below its steady-state before the genetic manipulations. The developed framework is demonstrated by studying the overproduction of ethanol in Escherichia coli. Computational results reveal the existence of synergism between reaction deletions and modulations implying that the simultaneous application of both types of genetic manipulations yields the most promising results. For example, the downregulation of phosphoglucomutase in conjunction with the deletion of oxygen uptake and pyruvate formate lyase yields 99.8% of the maximum theoretical ethanol yield. Conceptually, the proposed strategies redirect both the carbon flux as well as the cofactors to enhance ethanol production in the network. The OptReg framework is a versatile tool for strain design which allows for a broad array of genetic manipulations.     

Allostery and conformational free energy changes in human tryptophanyl‐tRNA synthetase from essential dynamics and structure networks

The interdependence of the concept of allostery and enzymatic catalysis, and they being guided by conformational mobility is gaining increased prominence. However, to gain a molecular level understanding of allostery and hence of enzymatic catalysis, it is of utter importance that the networks of amino acids participating in allostery be deciphered. Our lab has been exploring the methods of network analysis combined with molecular dynamics simulations to understand allostery at molecular level. Earlier we had outlined methods to obtain communication paths and then to map the rigid/flexible regions of proteins through network parameters like the shortest correlated paths, cliques, and communities. In this article, we advance the methodology to estimate the conformational populations in terms of cliques/communities formed by interactions including the side‐chains and then to compute the ligand‐induced population shift. Finally, we obtain the free‐energy landscape of the protein in equilibrium, characterizing the free‐energy minima accessed by the protein complexes. We have chosen human tryptophanyl‐tRNA synthetase (hTrpRS), a protein responsible for charging tryptophan to its cognate tRNA during protein biosynthesis for this investigation. This is a multidomain protein exhibiting excellent allosteric communication. Our approach has provided valuable structural as well as functional insights into the protein. The methodology adopted here is highly generalized to illuminate the linkage between protein structure networks and conformational mobility involved in the allosteric mechanism in any protein with known structure. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Synthesis of novel 3-amino and 29-hydroxamic acid derivatives of glycyrrhetinic acid as selective 11β-hydroxysteroid dehydrogenase 2 inhibitors

Glycyrrhetinic acid, the metabolite of the natural product glycyrrhizin, is a well known nonselective inhibitor of 11β-hydroxysteroid dehydrogenase (11β-HSD) type 1 and type 2. Whereas inhibition of 11β-HSD1 is currently under consideration for treatment of metabolic diseases, such as obesity and diabetes, 11β-HSD2 inhibitors may find therapeutic applications in chronic inflammatory diseases and certain forms of cancer. So far, no selective 11β-HSD2 inhibitor has been developed and neither animal studies nor clinical trials have been reported based on 11β-HSD2 inhibition. Starting from the lead compound glycyrrhetinic acid, novel triterpene type derivatives were synthesized and analyzed for their biological activity against overexpressed human 11β-HSD1 and 11β-HSD2 in cell lysates. Several hydroxamic acid derivatives showed high selectivity for 11β-HSD2. The most potent and selective compound is active against human 11β-HSD2 in the low nanomolar range with a 350-fold selectivity over human 11β-HSD1.     

Biodegradation of feather keratin with a PEGylated protease of Chryseobacterium gleum

Present study deals with the covalent modification of keratinolytic protease of Chryseobacterium gleum with higher enzyme activity, improved stability, non-immunogenicity and reusability. Protease of C. gleum showing feather degradation ability was modified by covalent attachment to polyethylene glycol. This modification culminated the change in electrophoretic mobility of protease in acrylamide gel. The modified enzyme showed 1.4 times more catalytic activity with better stability than native in aqueous system containing whole feathers as keratin. It showed improved pH, thermal, storage and solvent stability with a broadened range of pH (7–9) and temperature (25–50 °C) than native. The differentiation between modified and native enzyme was authenticated through UV–vis spectroscopy, SEM, XRD, FTIR and DSC. This modification of protease proved to be non-immunogenic in rats. The enzyme extracted after first run could be used for several cycles which clearly demonstrated its reusability in catalytic bioprocess of keratin degradation.     

Adaptation of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> to increased NaCl concentration due to dominant beneficial mutations

New beneficial mutations, combined with selection, were responsible for quick adaptation of Drosophila melanogaster to a novel environment. Using a highly inbred homozygous stock of D. melanogaster, we observed that in thirty generations the original stock had evolved to resist a previously toxic level of dietary salt (NaCl) and to produce a significantly higher number of progeny when reared in elevated salt concentrations. Survival in higher salt-stressed environments was due to new dominant genetic changes on the second and third chromosomes.     

Expression, purification, and characterisation of nesiritide using an E. coli expression system

Nesiritide, the recombinant human brain natriuretic peptide, is involved in the regulation of cardiovascular homeostasis and has been approved for treatment of patients with congestive heart failure. We prepared a synthetic cDNA construct of Nesiritide to generate a fusion protein with an affinity handle and 41 amino acid peptide of β-galactosidase. The fusion protein was expressed mainly in the inclusion bodies and accounted for approximately 20% of total cellular protein. After purification by Ni-IDA affinity chromatography and renaturation, the fusion protein was cleaved with purified recombinant enterokinase. Nesiritide was purified by pH precipitation/ion exchange chromatography followed by source phenyl chromatography to obtain protein with > 99% purity (determined by RPHPLC) and a mass of 3,464 Daltons. The potency (ED₅₀) of the purified protein was equivalent to that of Natrecor (Innovator formulation). Analytical methods were developed to identify oxidised, reduced and other related impurities. The expression strategy described in this work allows the convenient generation of high yield Nesiritide and enabled ease of purification.     

The bladder tumor suppressor protein TERE1 (UBIAD1) modulates cell cholesterol: implications for tumor progression

Convergent evidence implicates the TERE1 protein in human bladder tumor progression and lipid metabolism. Previously, reduced TERE1 expression was found in invasive urologic cancers and inhibited cell growth upon re-expression. A role in lipid metabolism was suggested by TERE1 binding to APOE, a cholesterol carrier, and to TBL2, a candidate protein in triglyceride disorders. Natural TERE1 mutations associate with Schnyder's corneal dystrophy, characterized by lipid accumulation. TERE1 catalyzes menaquinone synthesis, known to affect cholesterol homeostasis. To explore this relationship, we altered TERE1 and TBL2 dosage via ectopic expression and interfering RNA and measured cholesterol by Amplex red. Protein interactions of wild-type and mutant TERE1 with GST-APOE were evaluated by binding assays and molecular modeling. We conducted a bladder tumor microarray TERE1 expression analysis and assayed tumorigenicity of J82 cells ectopically expressing TERE1. TERE1 expression was reduced in a third of invasive specimens. Ectopic TERE1 expression in J82 bladder cancer cells dramatically inhibited nude mouse tumorigenesis. TERE1 and TBL2 proteins inversely modulated cellular cholesterol in HEK293 and bladder cancer cells from 20% to 50%. TERE1 point mutations affected APOE interactions, and resulted in cholesterol levels that differed from wild type. Elevated tumor cell cholesterol is known to affect apoptosis and growth signaling; thus, loss of TERE1 in invasive bladder cancer may represent a defect in menaquinone-mediated cholesterol homeostasis that contributes to progression.