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Papers by sukumar adak

Journal of Biological Chemistry, Mar 31, 2000

A ferrous heme-NO complex builds up in rat neuronal NO synthase during catalysis and lowers its a... more A ferrous heme-NO complex builds up in rat neuronal NO synthase during catalysis and lowers its activity. Mutation of a tryptophan located directly below the heme (Trp 409) to Phe or Tyr causes hyperactive NO synthesis and less heme-NO complex buildup in the steady state (

Nitric-oxide synthases (NOSs) are widely distributed among prokaryotes and eukaryotes and have di... more Nitric-oxide synthases (NOSs) are widely distributed among prokaryotes and eukaryotes and have diverse functions in physiology. Recent genome sequencing revealed NOS-like protein in bacteria, but whether these proteins generate nitric oxide is unknown. We therefore cloned, expressed, and purified a NOS-like protein from Bacillus subtilis (bsNOS) and characterized its catalytic parameters in both multiple and single turnover reactions. bsNOS was dimeric, bound L-Arg and 6R-tetrahydrobiopterin with similar affinity as mammalian NOS, and generated nitrite from L-Arg when incubated with NADPH and a mammalian NOS reductase domain. Stopped-flow analysis showed that ferrous bsNOS reacted with O 2 to form a transient heme Fe(II)O 2 species in the presence of either Arg or the reaction intermediate N-hydroxy-L-arginine. In the latter case, disappearance of the Fe(II)O 2 species was kinetically and quantitatively coupled to formation of a transient heme Fe(III)NO product, which then dissociated to form ferric bsNOS. This behavior mirrors mammalian NOS enzymes and unambiguously shows that bsNOS can generate NO. NO formation required a bound tetrahydropteridine, and the kinetic effects of this cofactor were consistent with it donating an electron to the Fe(II)O 2 intermediate during the reaction. Dissociation of the heme Fe(III)NO product was much slower in bsNOS than in mammalian NOS. This constrains allowable rates of ferric heme reduction by a protein redox partner and underscores the utility of using a tetrahydropteridine electron donor in bsNOS. Nitric-oxide synthases (NOSs, EC 1.14.13.39) 1 are present in insects, mollusks, parasites, fungi, slime molds, and bacteria (1-4). Their amino acid sequences and activities are similar to the mammalian NOSs, suggesting that the mammalian gene came from lower species through evolution. The mammalian NOSs catalyze the oxidation of L-arginine (Arg) to citrulline 2 Preliminary sequence data were obtained from The Institute for Genomic Research website at www.tigr.org.

$Research paper thumbnail of Preparation and characterization of an Al 2O 3–ZrO 2 nanocomposite, Part I: Powder synthesis and transformation behavior during fracture$

Compos Part a Appl Sci Manuf, 2007

ABSTRACT

Ceramics International, 2005

Zirconia dispersed alumina powders have been synthesized in homogenous condition by following the... more Zirconia dispersed alumina powders have been synthesized in homogenous condition by following the wet interaction process at varying ZrO 2 content and the thermo-mechanical behavior of the same has been studied. The tetragonal phase is retained at room temperature without any deliberate addition of stabilizer, which has been explained on the basis of particle size effect in terms of lower surface energy of the t-phase compensating for the difference in chemical free energy. Flexural strength follows an inverse relation with temperature due to reduction of driving force for t ! m transformation. The toughness and strength reduce significantly above 10 mol.% ZrO 2 beyond which comparatively large grain growth is observed. The thermal expansion hysteresis associated with t ! m transformation is evident with martensitic temperature influenced by ZrO 2 content and grain size.

Journal of Biological Chemistry, 2001

Journal of Materials Science

... Nanostructured Al2O3ZrO2 composite synthesized by solgel technique: powder processing and m... more ... Nanostructured Al2O3ZrO2 composite synthesized by solgel technique: powder processing and microstructure Debasish Sarkar Æ Deepak Mohapatra Æ Sambarta Ray Æ Santanu Bhattacharyya Æ Sukumar Adak Æ Niren Mitra ...

Journal of Biological Chemistry

The heme of neuronal nitric-oxide synthase participates in oxygen activation but also binds self-... more The heme of neuronal nitric-oxide synthase participates in oxygen activation but also binds self-generated NO during catalysis resulting in reversible feedback inhibition. We utilized point mutagenesis to investigate if a conserved tryptophan residue (Trp-409), which engages in-stacking with the heme and hydrogen bonds to its axial cysteine ligand, helps control catalysis and regulation by NO. Surprisingly, mutants W409F and W409Y were hyperactive compared with the wild type regarding NO synthesis without affecting cytochrome c reduction, reductase-independent N-hydroxyarginine oxidation, or Arg and tetrahydrobiopterin binding. In the absence of Arg, NADPH oxidation measurements showed that electron flux through the heme was actually slower in the Trp-409 mutants than in wild-type nNOS. However, little or no NO complex accumulated during NO synthesis by the mutants, as opposed to the wild type. This difference was potentially related to mutants forming unstable 6-coordinate ferrous-NO complexes under anaerobic conditions even in the presence of Arg and tetrahydrobiopterin. Thus, Trp-409 mutations minimize NO feedback inhibition by preventing buildup of an inactive ferrous-NO complex during the steady state. This overcomes the negative effect of the mutation on electron flux and results in hyperactivity. Conservation of Trp-409 among different NOS suggests that the ability of this residue to regulate heme reduction and NO complex formation is important for enzyme physiologic function.

Journal of Biological Chemistry

We studied catalysis by tetrahydrobiopterin (H4B)free neuronal nitric-oxide synthase (nNOS) to un... more We studied catalysis by tetrahydrobiopterin (H4B)free neuronal nitric-oxide synthase (nNOS) to understand how heme and H4B participate in nitric oxide (NO) synthesis. H4B-free nNOS catalyzed Arg oxidation to N-hydroxy-L-Arg (NOHA) and citrulline in both NADPH-and H 2 O 2-driven reactions. Citrulline formation was time-and enzyme concentration-dependent but was uncoupled relative to NADPH oxidation, and generated nitrite and nitrate without forming NO. Similar results were observed when NOHA served as substrate. Steady-state and stopped-flow spectroscopy with the H4B-free enzyme revealed that a ferrous heme-NO complex built up after initiating catalysis in both NADPHand H 2 O 2-driven reactions, consistent with formation of nitroxyl as an immediate product. This differed from the H4B-replete enzyme, which formed a ferric heme-NO complex as an immediate product that could then release NO. We make the following conclusions. 1) H4B is not essential for Arg oxidation by nNOS, although it helps couple NADPH oxidation to product formation in both steps of NO synthesis. Thus, the NADPH-or H 2 O 2driven reactions form common heme-oxy species that can react with substrate in the presence or absence of H4B. 2) The sole essential role of H4B is to enable nNOS to generate NO instead of nitroxyl. On this basis we propose a new unified model for heme-dependent oxygen activation and H4B function in both steps of NO synthesis.

$Research paper thumbnail of Study on low carbon containing MgO-C refractory: Use of nano carbon$

Ceramics International

Development of nano carbon containing magnesia carbon refractories has been studied to reduce the... more Development of nano carbon containing magnesia carbon refractories has been studied to reduce the total carbon content, thereby reducing the heat loss from the metallurgical process and producing more eco-friendly refractories. The carbon contamination from refractory to liquid metal will be minimized using low amount of nano carbon. Different percentages of nano carbon are used in combination with graphite as carbon source and the total carbon is maintained below the half of the total carbon of the conventional MgO-C refractories. The compositions were processed as per the conventional manufacturing techniques and the properties were evaluated and compared against the conventional refractory prepared under exactly similar conditions. Also elemental mapping of carbon was done to study the distribution of the nano carbon in the matrix.

Microstructural evolution and grain growth in 3Y-TZP ceramics have been examined using two nano-s... more Microstructural evolution and grain growth in 3Y-TZP ceramics have been examined using two nano-sized 3Y-TZP powders, calcined at different temperatures, derived from the same hydroxide powder. The green bodies prepared from both powders showed two sets of porosity: inter-agglomerate and intra-agglomerate. The intra-agglomerate pores are related to initial crystallite size; the size of the inter-agglomerate pores are governed by the pressure applied to the green bodies. Due to the nano-sized dimensions, the intra-agglomerate porosity has little effect on the final density and grain size. In contrast, the large inter-agglomerate pores play a major role on both the sintering process and achievement of final density. During sintering, the grains initially grew to approximately 100 nm, reaching the intermediate stage of sintering; the grain growth rate then became very much reduced before reaching the final stage of the sintering process. At this point, significant grain growth took place, possibly controlled by the solution drag mechanism. The activation energy for the grain growth was determined to be 352 kJ/mol.

The nNOS reductase domain is homologous to cytochrome P450 reductase, which contains two conserve... more The nNOS reductase domain is homologous to cytochrome P450 reductase, which contains two conserved clusters of acidic residues in its FMN module that play varied roles in its electron transfer reactions. To study the role of nNOS reductase domain cluster 1 acidic residues, we mutated two conserved acidic (Asp 918 and Glu 919) and one conserved aromatic residue (Phe 892), and investigated the effect of each mutation on flavin binding, conformational change, electron transfer reactions, calmodulin regulation, and catalytic activities. Each mutation destabilized FMN binding without significantly affecting other aspects including substrate, cofactor or calmodulin binding, or catalytic activities upon FMN reconstitution, indicating the mutational effect was restricted to the FMN module. Characterization of the FMN-depleted mutants showed that bound FMN was essential for reduction of the nNOS heme or cytochrome c, but not for ferricyanide or dichlorophenolindolphenol, and established that the electron transfer path in nNOS is NADPH to FAD to FMN to heme. Steadystate and stopped-flow kinetic analysis revealed a novel role for bound FMN in suppressing FAD reduction by NADPH. The suppression could be relieved either by FMN removal or calmodulin binding. Calmodulin binding induced a conformational change that was restricted to the FMN module. This increased the rate of FMN reduction and triggered electron transfer to the heme. We propose that the FMN module of nNOS is the key positive or negative regulator of electron transfer at all points in nNOS. This distinguishes nNOS from other related flavoproteins, and helps explain the mechanism of calmodulin regulation.

$Research paper thumbnail of Influence of Nanocrystalline MgAl 2 O 4 Spinel Addition on the Properties of MgO-C Refractories$

Materials and Manufacturing Processes, 2012

... 6. Soboyejo , WO ; Akpan , ET ; Bashir , IB ; Zimba , J. ; Hosannah , N. ; Allameh , S. Effec... more ... 6. Soboyejo , WO ; Akpan , ET ; Bashir , IB ; Zimba , J. ; Hosannah , N. ; Allameh , S. Effects of Na 2 O on the thermal shock resistance of ... Mukhopadhyay , S. ; Pal , P. ; Nag , B. ; Jana , P. Influence of gel-derived nanocrystalline spinel in a high alumina castable: Part 2 . Ceramic ...

Journal of Materials Science, 2007