Papers by amlan roychowdhury
Nanomaterials in Clinical Therapeutics
Nucleic Acids Research, Aug 22, 2019
Ribosome biogenesis is an essential process in all living cells, which entails countless highly s... more Ribosome biogenesis is an essential process in all living cells, which entails countless highly sequential and dynamic structural reorganization events. These include formation of dozens RNA helices through Watson-Crick base-pairing within ribosomal RNAs (rRNAs) and between rRNAs and small nucleolar RNAs (snoRNAs), transient association of hundreds of proteinaceous assembly factors to nascent precursor (pre-)ribosomes, and stable assembly of ribosomal proteins. Unsurprisingly, the largest group of ribosome assembly factors are energy-consuming proteins (NTPases) including 25 RNA helicases in budding yeast. Among these, the DEAH-box Dhr1 is essential to displace the box C/D snoRNA U3 from the pre-rRNAs where it is bound in order to prevent premature formation of the central pseudoknot, a dramatic irreversible long-range interaction essential to the overall folding of the small ribosomal subunit. Here, we report the crystal structure of the Dhr1 helicase module, revealing the presence...
Biochimie, 2019
The redox homeostasis of cytoplasm is maintained by a series of disulfide exchange reactions medi... more The redox homeostasis of cytoplasm is maintained by a series of disulfide exchange reactions mediated by proteins belonging to the thioredoxin superfamily. Thioredoxin and thioredoxin reductase, being the major members of the family, play a key role in oxidative stress response of Staphylococcus aureus. In this report, we have identified and characterised an active thioredoxin system of the mentioned pathogen. Crystal structure of thioredoxin2 (SaTrx2) in its reduced form reveals that it contains the conserved redox active WCXXC motif and a thioredoxin fold. Thioredoxin reductase2 (SaTR2) is a flavoprotein and consists of two Rossmann folds as the binding sites for FAD and NADPH. Crystal structure of the SaTR2 holoenzyme shows that the protein consists of two domains and the catalytic site comprises of an intramolecular disulfide bond formed between two sequentially distal cysteine residues. Biophysical and biochemical studies unveil that SaTrx2 and SaTR2 can physically interact in solution and in the course of sustaining the redox equilibrium, the latter reduces the former. Molecular docking has been performed to illustrate the interface formed between SaTrx2 and SaTR2 during the disulfide exchange reaction.
Acta Crystallographica Section F Structural Biology and Crystallization Communications, Jun 1, 2011
The cysteine-based protein phosphatase H1L was the first reported dualspecificity protein phospha... more The cysteine-based protein phosphatase H1L was the first reported dualspecificity protein phosphatase. H1L is encapsidated within the vaccinia virus and is required for successful host infection and for the production of viable vaccinia progeny. H1L has therefore been proposed as a target candidate for antiviral compounds. Recombinant H1L has been expressed in a catalytically inactive form using an Escherichia coli host, leading to purification and crystallization by the microbatch method. The crystals diffract to 2.1 Å resolution using synchrotron radiation. These crystals belong to space group P422, with unit-cell parameters a = b = 98.31, c = 169.15 Å , and are likely to contain four molecules in the asymmetric unit. A sulfur SAD data set was collected to 2.8 Å resolution on beamline BM14 at the ESRF to facilitate structure determination. Attempts to derivatize these crystals with xenon gas changed the space group to I422, with unit-cell parameters a = b = 63.28, c = 169.68 Å and a single molecule in the asymmetric unit. The relationship between these two crystal forms is discussed.
Journal of General Virology, 2016
Antheraea mylitta cytoplasmic polyhedrosis virus is a segmented dsRNA virus of the family Reoviri... more Antheraea mylitta cytoplasmic polyhedrosis virus is a segmented dsRNA virus of the family Reoviridae. Segment 2 (S2)-encoded RNA-dependent RNA polymerase (RdRp) helps the virus to propagate its genome in the host cell of the silkworm, Antheraea mylitta. Cloning, expression, purification and functional analysis of individual domains of RdRp have demonstrated that the purified domains interact in vitro. The central polymerase domain (PD) shows nucleotide binding properties, but neither the N-terminal domain (NTD) nor the Cterminal domain (CTD). Isolated PD does not exhibit RdRp activity but this activity can be reconstituted when all three domains are included in the reaction mixture. Molecular dynamics simulation suggests that the isolated PD has increased internal motions in comparison to when it is associated with the NTD and CTD. The motions of the separated PD may lead to the formation of a less accessible RNA template-binding channel and, thus, impair RdRp activity.
Acta Crystallographica Section F Structural Biology Communications, 2013
Histidine triad nucleotide-binding protein 2 (HINT2) is a mitochondrial adenosine phosphoramidase... more Histidine triad nucleotide-binding protein 2 (HINT2) is a mitochondrial adenosine phosphoramidase mainly expressed in the pancreas, liver and adrenal gland. HINT2 possibly plays a role in apoptosis, as well as being involved in steroid biosynthesis, hepatic lipid metabolism and regulation of hepatic mitochondria function. The expression level of HINT2 is significantly down-regulated in hepatocellular carcinoma patients. To date, endogenous substrates for this enzyme, as well as the three-dimensional structure of human HINT2, are unknown. In this study, human HINT2 was cloned, overexpressed in Escherichia coli and purified. Crystallization was performed at 278 K using PEG 4000 as the main precipitant; the crystals, which belonged to the tetragonal space group P41212 with unit-cell parameters a = b = 76.38, c = 133.25 Å, diffracted to 2.83 Å resolution. Assuming two molecules in the asymmetric unit, the Matthews coefficient and the solvent content were calculated to be 2.63 Å(3) Da(-1) and 53.27%, respectively.
Acta crystallographica. Section F, Structural biology communications, 2014
Phosphoglycerate mutase (PGM) is a key enzyme in carbohydrate metabolism. It takes part in both g... more Phosphoglycerate mutase (PGM) is a key enzyme in carbohydrate metabolism. It takes part in both glycolysis and gluconeogenesis. PGM from pathogenic Staphylococcus aureus (NCTC8325) was cloned in pQE30 expression vector overexpressed in Escherichia coli M15 (pREP4) cells and purified to homogeneity. The protein was crystallized from two different conditions, (i) 0.1 M HEPES pH 7.5, 20%(w/v) polyethylene glycol 10,000 and (ii) 0.2 M NaCl, 0.1 M bis-tris pH 6.5, 25%(w/v) polyethylene glycol 3350, at 25°C by the sitting-drop vapour-diffusion method. Crystals grown at pH 7.5 diffracted to 2.5 Å resolution and belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 77.0, b = 86.11, c = 94.07 Å. Crystals from the second condition at pH 6.5 diffracted to 2.00 Å resolution. These crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 73.21, b = 81.75, c = 89.18 Å. X-ray diffraction data have been collected and processed to th...
Biochemical Journal, 2013
FabGs, or β-oxoacyl reductases, are involved in fatty acid synthesis. The reaction entails NADPH/... more FabGs, or β-oxoacyl reductases, are involved in fatty acid synthesis. The reaction entails NADPH/NADH-mediated conversion of β-oxoacyl-ACP (acyl-carrier protein) into β-hydroxyacyl-ACP. HMwFabGs (high-molecular-weight FabG) form a phylogenetically separate group of FabG enzymes. FabG4, an HMwFabG from Mycobacterium tuberculosis, contains two distinct domains, an N-terminal ‘flavodoxintype’ domain and a C-terminal oxoreductase domain. The catalytically active C-terminal domain utilizes NADH to reduce β-oxoacyl-CoA to β-hydroxyacyl-CoA. In the present study the crystal structures of the FabG4–NADH binary complex and the FabG4–NAD+–hexanoyl-CoA ternary complex have been determined to understand the substrate specificity and catalytic mechanism of FabG4. This is the first report to demonstrate how FabG4 interacts with its coenzyme NADH and hexanoyl-CoA that mimics an elongating fattyacyl chain covalently linked with CoA. Structural analysis shows that the binding of hexanoyl-CoA within ...
Cofactor‐independent phosphoglycerate mutase (iPGM), an important enzyme in glycolysis and glucon... more Cofactor‐independent phosphoglycerate mutase (iPGM), an important enzyme in glycolysis and gluconeogenesis, catalyses the isomerization of 2‐ and 3‐phosphoglycerates by an Mn2+‐dependent phospho‐transfer mechanism via a phospho‐enzyme intermediate. Crystal structures of bi‐domain iPGM from Staphylococcus aureus, together with substrate‐bound forms, have revealed a new conformation of the enzyme, representing an intermediate state of domain movement. The substrate‐binding site and the catalytic site are present in two distinct domains in the intermediate form. X–ray crystallography complemented by simulated dynamics has enabled delineation of the complete catalytic cycle, which includes binding of the substrate, followed by its positioning into the catalytic site, phospho‐transfer and finally product release. The present work describes a novel mechanism of domain movement controlled by a hydrophobic patch that is exposed on domain closure and acts like a spring to keep the protein in...
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Papers by amlan roychowdhury