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Yadav S.,National Institute of Plant Genome Research | Kushwaha H.R.,Jawaharlal Nehru University | Kushwaha H.R.,Synthetic Biology and Biofuel Group | Kumar K.,National Institute of Plant Genome Research | Verma P.K.,National Institute of Plant Genome Research
International Journal of Biological Macromolecules | Year: 2012

Glutaredoxins (GRXs) are small, ubiquitous, multifunctional, heat-stable and glutathione-dependent thiol-disulphide oxidoreductases, classified under thioredoxin-fold superfamily. In the green lineage, GRXs constitute a complex family of proteins. Based on their active site, GRXs are classified into two subfamilies: dithiol and monothiol. Monothiol GRXs contain 'CGFS' as a redox active motif and assist in maintaining redox state and iron homeostasis within the cell. Using RACE strategy, a full length cDNA of chickpea (Cicer arietinum) glutaredoxin 3 (CarGRX3) was cloned and sequenced. The cDNA contains open reading frame of 537bp encoding 178 amino acids and exhibits features of other known 'CGFS' type GRXs. Based on the multiple sequence alignment among CarGRX3 and monothiol GRXs of other photosynthetic organisms, the characteristic motif (KGX4PXCGFSX[29/30/32]KX4WPTXPQX4GX3GGXDI) with 18 invariant residues was observed. The proposed structure of CarGRX3 was compared with structurally resolved monothiol GRXs of other organisms. The CarGRX3 and nearest Arabidopsis homolog (AtGRXcp) shares 76% sequence identity which was reflected by their 3D-structure conservation. The structure of chickpea monothiol GRX (CarGRX3) coordinates glutathione ligated [2Fe-2S] cluster in a homodimeric form, highlighting the structural basis for iron-sulfur cluster (ISC) assembly and delivery to acceptor proteins. The present study on CarGRX3 model highlighted the utility of the theoretical approaches to understand complex biological phenomena such as glutathione docking and incorporation of GSH-ligated [2Fe-2S] cluster. © 2012 Elsevier B.V.

Gaur N.A.,U.S. National Institutes of Health | Gaur N.A.,Synthetic Biology and Biofuel Group | Hasek J.,Academy of Sciences of the Czech Republic | Brickner D.G.,Northwestern University | And 8 more authors.
Genetics | Year: 2013

There is increasing evidence that certain Vacuolar protein sorting (Vps) proteins, factors that mediate vesicular protein trafficking, have additional roles in regulating transcription factors at the endosome. We found that yeast mutants lacking the phosphatidylinositol 3-phosphate [PI(3)P] kinase Vps34 or its associated protein kinase Vps15 display multiple phenotypes indicating impaired transcription elongation. These phenotypes include reduced mRNA production from long or G+C-rich coding sequences (CDS) without affecting the associated GAL1 promoter activity, and a reduced rate of RNA polymerase II (Pol II) progression through lacZ CDS in vivo. Consistent with reported genetic interactions with mutations affecting the histone acetyltransferase complex NuA4, vps15Δ and vps34Δ mutations reduce NuA4 occupancy in certain transcribed CDS. vps15Δ and vps34Δ mutants also exhibit impaired localization of the induced GAL1 gene to the nuclear periphery. We found unexpectedly that, similar to known transcription elongation factors, these and several other Vps factors can be cross-linked to the CDS of genes induced by Gcn4 or Gal4 in a manner dependent on transcriptional induction and stimulated by Cdk7/Kin28-dependent phosphorylation of the Pol II C-terminal domain (CTD). We also observed colocalization of a fraction of Vps15-GFP and Vps34-GFP with nuclear pores at nucleus-vacuole (NV) junctions in live cells. These findings suggest that Vps factors enhance the efficiency of transcription elongation in a manner involving their physical proximity to nuclear pores and transcribed chromatin. © 2013 by the Genetics Society of America.

Turan S.,Synthetic Biology and Biofuel Group | Cornish K.,Ohio State University | Kumar S.,Synthetic Biology and Biofuel Group
Australian Journal of Crop Science | Year: 2012

Salinity stress limits crop yield affecting plant growth and restricting the use of land. As world population is increasing at alarming rate, agricultural land is shrinking due to industrialization and/or habitat use. Hence, there is a need to utilize salt affected land to meet the food requirement. Although some success has been achieved through conventional breeding but its use is limited due to reproductive barrier and scarcity of genetic variations among major crops. The genetic engineering has proven a revolutionary technique to generate salt tolerant plants as one can transfer desired gene from any genetic resource and/or alter the expression of existing gene(s). There are examples of improved salinity tolerance in various crop plants through the use of genetic engineering. However, there is a further need of improvement for successful release of salt tolerant cultivars at field level. In this review, we have given a detailed update on production of salt-tolerant plants through genetic engineering. Future prospects and concerns, along with the importance of novel techniques, as well as plant breeding are also discussed.

Mattam A.J.,Synthetic Biology and Biofuel Group | Clomburg J.M.,Rice University | Gonzalez R.,Rice University | Yazdani S.S.,Synthetic Biology and Biofuel Group
Biotechnology Letters | Year: 2013

Glycerol has attracted the attention of scientific and industrial communities due to its generation in bulk quantities as a byproduct of biofuel industries. With the rapid growth of these industries in recent years, glycerol is frequently treated as a very low-value byproduct or even a waste product with a disposal cost associated to it. Glycerol is not only abundant and inexpensive but also can generate more reducing equivalents than glucose or xylose. This unique characteristic of glycerol offers a tremendous opportunity for its biological conversion to valuable products at higher yield. This review focuses on research efforts to utilize glycerol as a carbon source for the production of a variety of fuels and chemicals by both native and metabolically engineered microorganisms. © 2013 Springer Science+Business Media Dordrecht.

Adlakha N.,Synthetic Biology and Biofuel Group | Sawant S.,Institute of Chemical Technology | Anil A.,Institute of Chemical Technology | Lali A.,Institute of Chemical Technology | Yazdani S.S.,Synthetic Biology and Biofuel Group
Applied and Environmental Microbiology | Year: 2012

Identification and design of new cellulolytic enzymes with higher catalytic efficiency are a key factor in reducing the production cost of lignocellulosic bioalcohol. We report here identification of a novel β-glucosidase (Gluc1C) from Paenibacillus sp. strain MTCC 5639 and construction of bifunctional chimeric proteins based on Gluc1C and Endo5A, a β-1,4-endoglucanase isolated from MTCC 5639 earlier. The 448-amino-acid-long Gluc1C contained a GH superfamily 1 domain and hydrolyzed cellodextrin up to a five-sugar chain length, with highest efficiency toward cellobiose. Addition of Gluc1C improved the ability of Endo5A to release the reducing sugars from carboxymethyl cellulose. We therefore constructed six bifunctional chimeric proteins based on Endo5A and Gluc1C varying in the positions and sizes of linkers. One of the constructs, EG5, consisting of Endo5A-(G4S)3-Gluc1C, demonstrated 3.2- and 2-fold higher molar specific activities for β-glucosidase and endoglucanase, respectively, than Gluc1C and Endo5A alone. EG5 also showed 2-fold higher catalytic efficiency than individual recombinant enzymes. The thermal denaturation monitored by circular dichroism (CD) spectroscopy demonstrated that the fusion of Gluc1C with Endo5A resulted in increased thermostability of both domains by 5oC and 9oC, respectively. Comparative hydrolysis experiments done on alkali-treated rice straw and CMC indicated 2-fold higher release of product by EG5 than that by the physical mixture of Endo5A and Gluc1C, providing a rationale for channeling of intermediates. Addition of EG5 to a commercial enzyme preparation significantly enhanced release of reducing sugars from pretreated biomass, indicating its commercial applicability. © 2012, American Society for Microbiology.

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