Debajyoti Dutta

Hydrogen production by Cyanobacteria

The limited fossil fuel prompts the prospecting of various unconventional energy sources to take over the traditional fossil fuel energy source. In this respect the use of hydrogen gas is an attractive alternate source. Attributed by its numerous advantages including those of environmentally clean, efficiency and renew ability, hydrogen gas is considered to be one of the most desired alternate. Cyanobacteria are highly promising microorganism for hydrogen production. In comparison to the traditional ways of hydrogen production (chemical, photoelectrical), Cyanobacterial hydrogen production is commercially viable. This review highlights the basic biology of cynobacterial hydrogen production, strains involved, large-scale hydrogen production and its future prospects. While integrating the existing knowledge and technology, much future improvement and progress is to be done before hydrogen is accepted as a commercial primary energy source.

Crystal structure and fluorescence studies reveal the role of helical dimeric interface of staphylococcal fabg1 in positive cooperativity for NADPH

Crystal structure of Staphylococcal β‐ketoacyl‐ACP reductase 1 (SaFabG1) complexed with NADPH is determined at 2.5 Å resolution. The enzyme is essential in FAS‐II pathway and utilizes NADPH to reduce β‐ketoacyl‐ACP to (S)‐β‐hydroxyacyl‐ACP. Unlike the tetrameric FabGs, dimeric SaFabG1 shows positive homotropic cooperativity towards NADPH. Analysis of FabG:NADPH binary crystal structure endorses that NADPH interacts directly with the helices α4 and α5 those are present on a dimerization interface. A steady shift in tryptophan (of α4 helix) emission peak upon steady increment of NADPH concentration reveals that the dimeric interface is formed by α4‐α4′ and α5‐α5′ helices. This dimeric interface imparts positive homotropic cooperativity towards NADPH. PEG, a substrate mimicking molecule is also found near the active site of the enzyme. Proteins 2012;

Design, synthesis and inhibition activity of novel cyclic peptides against protein tyrosine phosphatase A from Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent for tuberculosis has employed several signalling molecules to sense the host cellular environment and act accordingly. For example, protein tyrosine phosphatase A (MPtpA) of M. tuberculosis, a signalling protein belonging to the tyrosine phosphatase superfamily, is involved in phagocytosis and is active in virulent mycobacterial form. Starting from a β-lactam framework a new class of structure based cyclic peptide (CP) inhibitors was designed. The synthesis involves a crucial intramolecular transamidation via a ring opening reaction. All the compounds show moderate to good inhibitory activities against MPtpA in micromolar concentrations. The results of inhibition kinetics suggest mixed mode of inhibition. The binding constant determined from circular dichroism (CD) and fluorescence quenching studies shows strong binding of the hydrophilic side chain of CPs with the enzyme active site residues. All these are well supported by docking studies.

Scabin, a Novel DNA-acting ADP-ribosyltransferase from Streptomyces scabies

A bioinformatics strategy was used to identify Scabin, a novel DNA-targeting enzyme from the plant pathogen 87.22 strain of Streptomyces scabies. Scabin shares nearly 40% sequence identity with the Pierisin family of mono-ADP-ribosyltransferase toxins. Scabin was purified to homogeneity as a 22-kDa single-domain enzyme and was shown to possess high NAD+-glycohydrolase (Km(NAD) = 68 ± 3 μm; kcat = 94 ± 2 min−1) activity with an RSQXE motif; it was also shown to target deoxyguanosine and showed sigmoidal enzyme kinetics (K0.5(deoxyguanosine) = 302 ± 12 μm; kcat = 14 min−1). Mass spectrometry analysis revealed that Scabin labels the exocyclic amino group on guanine bases in either single-stranded or double-stranded DNA. Several small molecule inhibitors were identified, and the most potent compounds were found to inhibit the enzyme activity with Ki values ranging from 3 to 24 μm. PJ34, a well known inhibitor of poly-ADP-ribosyltransferases, was shown to be the most potent inhibitor of Scabin. Scabin was crystallized, representing the first structure of a DNA-targeting mono-ADP-ribosyltransferase enzyme; the structures of the apo-form (1.45 Å) and with two inhibitors (P6-E, 1.4 Å; PJ34, 1.6 Å) were solved. These x-ray structures are also the first high resolution structures of the Pierisin subgroup of the mono-ADP-ribosyltransferase toxin family. A model of Scabin with its DNA substrate is also proposed.

Structural elucidation of the binding site and mode of inhibition of Li+ and Mg2+ in inositol monophosphatase

Mg2+‐dependent, Li+‐sensitive phosphatases are a widely distributed family of enzymes with significant importance throughout the biological kingdom. Inositol monophosphatase (IMPase) is an important target of Li+‐based therapeutic agents in manic depressive disorders. However, despite decades of intense research efforts, the precise mechanism of Li+‐induced inhibition of IMPase remains obscured. Here we describe a structural investigation of the Li+ binding site in staphylococcal IMPase I (SaIMPase I) using X–ray crystallography. The biochemical study indicated common or overlapping binding sites for Mg2+ and Li+ in the active site of SaIMPase I. The crystal structure of the SaIMPase I ternary product complex shows the presence of a phosphate and three Mg2+ ions (namely Mg1, Mg2 and Mg3) in the active site. As Li+ is virtually invisible in X–ray crystallography, competitive displacement of Mg2+ ions from the SaIMPase I ternary product complex as a function of increasing LiCl concentration was used to identify the Li+ binding site. In this approach, the disappearing electron density of Mg2+ ions due to Li+ ion binding was traced, and the Mg2+ ion present at the Mg2 binding site was found to be replaced. Moreover, based on a detailed comparative investigation of the phosphate orientation and coordination states of Mg2+ binding sites in enzyme–substrate and enzyme–product complexes, inhibition mechanisms for Li+ and Mg2+ are proposed.

Crystal structure of Staphylococcal dual specific inositol monophosphatase/NADP(H) phosphatase (SAS2203) delineates the molecular basis of substrate specificity.

Inositol monophosphatase (IMPase) family of proteins are Mg(2+) activated Li(+) inhibited class of ubiquitous enzymes with promiscuous substrate specificity. Herein, the molecular basis of IMPase substrate specificity is delineated by comparative crystal structural analysis of a Staphylococcal dual specific IMPase/NADP(H) phosphatase (SaIMPase - I) with other IMPases of different substrate compatibility, empowered by in silico docking and Escherichia coli SuhB mutagenesis analysis. Unlike its eubacterial and eukaryotic NADP(H) non-hydrolyzing counterparts, the composite structure of SaIMPase - I active site pocket exhibits high structural resemblance with archaeal NADP(H) hydrolyzing dual specific IMPase/FBPase. The large and shallow SaIMPase - I active site cleft efficiently accommodate large incoming substrates like NADP(H), and therefore, justifies the eminent NADP(H) phosphatase activity of SaIMPase - I. Compared to other NADP(H) non-hydrolyzing IMPases, the profound difference in active site topology as well as the unique NADP(H) recognition capability of SaIMPase - I stems from the differential length and orientation of a distant helix α4 (in human and bovine α5) and its preceding loop. We identified the length of α4 and its preceding loop as the most crucial factor that regulates IMPase substrate specificity by employing a size exclusion mechanism. Hence, in SaIMPase - I, the substrate promiscuity is a gain of function by trimming the length of α4 and its preceding loop, compared to other NADP(H) non-hydrolyzing IMPases. This study thus provides a biochemical - structural framework revealing the length and orientation of α4 and its preceding loop as the predisposing factor for the determination of IMPase substrate specificity.

Crystal structure of dehydratase component HadAB complex of mycobacterial FAS-II pathway.

Fatty acid biosynthesis type II in mycobacteria delivers the fatty acids required for mycolic acid synthesis. The pathway employs a unique maoC like β-hydroxyacyl-ACP dehydratase HadAB or HadBC heterodimer in the third step of the elongation cycle. Here we report the crystal structure of the HadAB complex determined using a Pb-SIRAS method. Crystal structure aided with enzymatic study establishes the roles of HadA as a scaffolding component and HadB as a catalytic component together indispensable for the activity. The detailed structural analysis of HadAB in combination with MD simulation endorses the spatial orientation of the central hot-dog helix and the dynamic nature of its associated loop in regulation of substrate specificities in dehydratase/hydratase family enzymes.

Structural variability of C3larvin toxin. Intrinsic dynamics of the α/β fold of the C3-like group of mono-ADP-ribosyltransferase toxins

C3larvin toxin is a new member of the C3 class of the mono-ADP-ribosyltransferase toxin family. The C3 toxins are known to covalently modify small G-proteins, e.g. RhoA, impairing their function, and serving as virulence factors for an offending pathogen. A full-length X-ray structure of C3larvin (2.3 Å) revealed that the characteristic mixed α/β fold consists of a central β-core flanked by two helical regions. Topologically, the protein can be separated into N and C lobes, each formed by a β-sheet and an α-motif, and connected by exposed loops involved in the recognition, binding, and catalysis of the toxin/enzyme, i.e. the ADP-ribosylation turn–turn and phosphate–nicotinamide PN loops. Herein, we provide two new C3larvin X-ray structures and present a systematic study of the toxin dynamics by first analyzing the experimental variability of the X-ray data-set followed by contrasting those results with theoretical predictions based on Elastic Network Models (GNM and ANM). We identify residues that participate in the stability of the N-lobe, putative hinges at loop residues, and energy-favored deformation vectors compatible with conformational changes of the key loops and 3D-subdomains (N/C-lobes), among the X-ray structures. We analyze a larger ensemble of known C3bot1 conformations and conclude that the characteristic ‘crab-claw’ movement may be driven by the main intrinsic modes of motion. Finally, via computational simulations, we identify harmonic and anharmonic fluctuations that might define the C3larvin ‘native state.’ Implications for docking protocols are derived.

Potential use of sugar binding proteins in reactors for regeneration of CO2 fixation acceptor D-Ribulose-1,5-bisphosphate.

Sugar binding proteins and binders of intermediate sugar metabolites derived from microbes are increasingly being used as reagents in new and expanding areas of biotechnology. The fixation of carbon dioxide at emission source has recently emerged as a technology with potentially significant implications for environmental biotechnology. Carbon dioxide is fixed onto a five carbon sugar D-ribulose-1,5-bisphosphate. We present a review of enzymatic and non-enzymatic binding proteins, for 3-phosphoglycerate (3PGA), 3-phosphoglyceraldehyde (3PGAL), dihydroxyacetone phosphate (DHAP), xylulose-5-phosphate (X5P) and ribulose-1,5-bisphosphate (RuBP) which could be potentially used in reactors regenerating RuBP from 3PGA. A series of reactors combined in a linear fashion has been previously shown to convert 3-PGA, (the product of fixed CO2 on RuBP as starting material) into RuBP (Bhattacharya et al., 2004; Bhattacharya, 2001). This was the basis for designing reactors harboring enzyme complexes/mixtures instead of linear combination of single-enzyme reactors for conversion of 3PGA into RuBP. Specific sugars in such enzyme-complex harboring reactors requires removal at key steps and fed to different reactors necessitating reversible sugar binders. In this review we present an account of existing microbial sugar binding proteins and their potential utility in these operations.

Expression and characterization of the SOS1 Arabidopsis salt tolerance protein

SOS1 is the plasma membrane Na+/H+ antiporter of Arabidopsis thaliana. It is responsible for the removal of intracellular sodium in exchange for an extracellular proton. SOS1 is composed of 1146 amino acids. Approximately 450 make the membrane domain, while the protein contains and a very large regulatory cytosolic domain of about 696 amino acids. Schizosaccharomyces pombe contains the salt tolerance Na+/H+ antiporter proteins sod2. We examined the ability of SOS1 to rescue salt tolerance in S. pombe with a knockout of the sod2 gene (sod2::ura4). In addition, we characterized the importance of the regulatory tail of SOS1, in expression of the protein in S. pombe. We expressed full-length SOS1 and SOS1 shortened at the C-terminus and ending at amino acids 766 (medium) and 481 (short). The short version of SOS1 conveyed salt tolerance to sod2::ura4 yeast and Western blotting revealed that the protein was present. The protein was also targeted to the plasma membrane. The medium and full-length SOS1 protein were partially degraded and were not as well expressed as the short version of SOS1. The SOS1 short protein was also able to reduce Na+ content in S. pombe. The full-length SOS1 dimerized and depended on the presence of the cytosolic tail. An analysis of SOS1 predicted a topology of 13 transmembrane segments, distinct from E. coli NhaA but similar to the Na+/H+ exchangers Methanocaldococcus jannaschii NhaP1 and Thermus thermophile NapA.