Rupam Sahoo

A novel cellulase free alkaliphilic xylanase from alkali tolerant Penicillium citrinum: production, purification and characterization

Aims:  The enzymatic hydrolysis of xylan has potential economic and environment‐friendly applications. Therefore, attention is focused here on the discovery of new extremophilic xylanase in order to meet the requirements of industry.

In vivo protein tyrosine nitration in S. cerevisiae: identification of tyrosine-nitrated proteins in mitochondria.

Protein tyrosine nitration (PTN) is a selective post-translational modification often associated with pathophysiological conditions. Although yeast cells lack of mammalian nitric oxide synthase (NOS) orthologues, still it has been shown that they are capable of producing nitric oxide (NO). Our studies showed that NO or reactive nitrogen species (RNS) produced in flavohemoglobin mutant (Deltayhb1) strain along with the wild type strain (Y190) of Saccharomyces cerevisiae can be visualized using specific probe 4,5-diaminofluorescein diacetate (DAF-2DA). Deltayhb1 strain of S. cerevisiae showed bright fluorescence under confocal microscope that proves NO or RNS accumulation is more in absence of flavohemoglobin. We further investigated PTN profile of both cytosol and mitochondria of Y190 and Deltayhb1 cells of S. cerevisiae using two-dimensional (2D) gel electrophoresis followed by western blot analysis. Surprisingly, we observed many immunopositive spots both in cytosol and in mitochondria from Y190 and Deltayhb1 using monoclonal anti-3-nitrotyrosine antibody indicating a basal level of NO or nitrite or peroxynitrite is produced in yeast system. To identify proteins nitrated in vivo we analyzed mitochondrial proteins from Y190 strains of S. cerevisiae. Among the eight identified proteins, two target mitochondrial proteins are aconitase and isocitrate dehydrogenase that are involved directly in the citric acid cycle. This investigation is the first comprehensive study to identify mitochondrial proteins nitrated in vivo.

A novel role of catalase in detoxification of peroxynitrite in S. cerevisiae.

The biological targets of peroxynitrite toxicity include wide array of biomolecules. Although several enzymes are found to be important components of cellular defense against peroxynitrite, the complete scenario is not totally understood. Yeast flavohemoglobin (YHB) and glutathione-dependent formaldehyde dehydrogenase (GS-FDH) confers resistance against nitric oxide and related reactive nitrogen species. In the present study, when subtoxic dose of peroxynitrite was applied to wild type, Deltayhb1 and Deltasfa1 strains of Saccharomyces cerevisiae, induction of cytosolic catalase was found at activity as well as gene expression level in mutants but not in wild type. Such induction was not due to intracellular reactive oxygen species (ROS) formation. Our in vitro studies confirmed the role of catalase in protection against peroxynitrite-mediated oxidation and nitration and also in peroxynitrite catabolism. This report is first of its kind regarding the novel role of catalase in peroxynitrite detoxification in Deltayhb1 and Deltasfa1 strains of S. cerevisiae.

Nitrosative stress on yeast: inhibition of glyoxalase-I and glyceraldehyde-3-phosphate dehydrogenase in the presence of GSNO.

Under nitrosative stressed condition intracellular GSNO accumulation is common to all cell types. Conserved NADH-dependent GSNO reductase was reported previously as an important cellular protective measure against this. In spite of the constitutive nature of the enzyme, we observed in vivo inactivation of two important enzymes-glyoxalase-I and glyceraldehyde-3-phosphate dehydrogenase under 5 mM GSNO stress in two budding yeasts, though with difference in their sensitivity. Former was more susceptible to inactivation in in vitro condition, too. In this study, we explored the competitive nature of yeast glyoxalase-I inhibition by GSNO. GSNO actually competes with GSH substrate-binding site of the enzyme.

Characterization of Drosophila nitric oxide synthase: a biochemical study

The heme and flavin-binding domains of Drosophila nitric oxide synthase (DNOS) were expressed in Escherichia coli using the expression vector pCW. The denatured molecular mass of the expressed protein was 152kDa along with a proteolytically cleaved product of 121kDa. The DNOS heme protein exhibited very low Ca(2+)/calmodulin-dependent NO synthase activity. The trypsin digestion patterns were different from nNOS. The full-length DNOS protein had high degree of stability against trypsin. The activity assay of trypsin-digested protein confirmed the same result. Urea dissociation profile of DNOS full-length protein showed that the reductase domain activity was much more susceptible towards urea than the oxygenase domain activity. Urea gradient gel of DNOS full-length protein established distinct transition of dissociation and unfolding in the range 3-4M urea. Reductase domain activity of full-length DNOS protein against external electron acceptors like cytochrome c indicated slow electron transfer from FMN. The bacterial expression of DNOS full-length protein represents an important development in structure-function studies of this enzyme and comparison with other mammalian NOS enzymes which is evolutionary significant.

Characterizing the effect of nitrosative stress in Saccharomyces cerevisiae

Nitrosative stress has various pathophysiological implications. We here present a detailed characterization on the effect of nitrosative stress in Saccharomyces cerevisiae wild-type (Y190) and its isogenic flavohemoglobin mutant (Deltayhb1) strain grown in presence of non fermentable carbon source. On addition of sub-toxic dose of nitrosating agent both the strains showed microbiostatic effect. Cellular respiration was found to be significantly affected in both the strains in presence sodium nitroprusside. Although there was no alteration in mitochondrial permeability potential changes and reactive oxygen species production in both the strains but the cellular redox status is differentially regulated in Deltayhb1 strain both in cytosol and in mitochondria indicating cellular glutathione is the major player in absence of flavohemoglobin. We also found important role(s) of various redox active enzymes like glutathione reductase and catalase in protection against nitrosative stress. This is the first report of its kind where the effect of nitrosative stress has been evaluated in S. cerevisiae cytosol as well as in mitochondria under respiratory proficient conditions.

Dissociation and unfolding of inducible nitric oxide synthase oxygenase domain identifies structural role of tetrahydrobiopterin in modulating the heme environment

The oxygenase domain of the inducible nitric oxide synthase, Δ65 iNOSox is a dimer that binds heme, L-Arginine (L-Arg), and tetrahydrobiopterin (H4B) and is the site for NO synthesis. The role of H4B in iNOS structure-function is complex and its exact structural role is presently unknown. The present paper provides a simple mechanistic account of interaction of the cofactor tetrahydrobiopterin (H4B) with the bacterially expressed Δ65 iNOSox protein. Transverse urea gradient gel electrophoresis studies indicated the presence of different conformers in the cofactor-incubated and cofactor-free Δ65 iNOSox protein. Dynamic Light Scattering (DLS) studies of cofactor-incubated and cofactor-free Δ65 iNOSox protein also showed two distinct populations of two different diameter ranges. Cofactor tetrahydrobiopterin (H4B) shifted one population, with higher diameter, to the lower diameter ranges indicating conformational changes. The additional role played by the cofactor is to elevate the heme retaining capacity even in presence of denaturing stress. Together, these findings confirm that the H4B is essential in modulating the iNOS heme environment and the protein environment in the dimeric iNOS oxygenase domain. (Mol Cell Boichem xxx: 1–10, 2005)