Dataset Information

Characterization of a Highly pH Stable Chi-Class Glutathione S-Transferase from Synechocystis PCC 6803.

ABSTRACT: Glutathione S-transferases (GSTs) are multifunctional enzymes present in virtually all organisms. Besides having an essential role in cellular detoxification, they also perform various other functions, including responses in stress conditions and signaling. GSTs are highly studied in plants and animals; however, the knowledge regarding GSTs in cyanobacteria seems rudimentary. In this study, we report the characterization of a highly pH stable GST from the model cyanobacterium--Synechocystis PCC 6803. The gene sll0067 was expressed in Escherichia coli (E. coli), and the protein was purified to homogeneity. The expressed protein exists as a homo-dimer, which is composed of about 20 kDa subunit. The results of the steady-state enzyme kinetics displayed protein's glutathione conjugation activity towards its class specific substrate- isothiocyanate, having the maximal activity with phenethyl isothiocyanate. Contrary to the poor catalytic activity and low specificity towards standard GST substrates such as 1-chloro-2,4-dinitrobenzene by bacterial GSTs, PmGST B1-1 from Proteus mirabilis, and E. coli GST, sll0067 has broad substrate degradation capability like most of the mammalian GST. Moreover, we have shown that cyanobacterial GST sll0067 is catalytically efficient compared to the best mammalian enzymes. The structural stability of GST was studied as a function of pH. The fluorescence and CD spectroscopy in combination with size exclusion chromatography showed a highly stable nature of the protein over a broad pH range from 2.0 to 11.0. To the best of our knowledge, this is the first GST with such a wide range of pH related structural stability. Furthermore, the presence of conserved Proline-53, structural motifs such as N-capping box and hydrophobic staple further aid in the stability and proper folding of cyanobacterial GST-sll0067.

SUBMITTER: Pandey T

PROVIDER: S-EPMC4429112 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Characterization of a Highly pH Stable Chi-Class Glutathione S-Transferase from Synechocystis PCC 6803.

Pandey Tripti T Singh Sudhir Kumar SK Chhetri Gaurav G Tripathi Timir T Singh Arvind Kumar AK

PloS one 20150512 5

Glutathione S-transferases (GSTs) are multifunctional enzymes present in virtually all organisms. Besides having an essential role in cellular detoxification, they also perform various other functions, including responses in stress conditions and signaling. GSTs are highly studied in plants and animals; however, the knowledge regarding GSTs in cyanobacteria seems rudimentary. In this study, we report the characterization of a highly pH stable GST from the model cyanobacterium--Synechocystis PCC ...[more]

PMID: 25965384

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Project description:In recent years, there has been an increased interest in the research and development of sustainable alternatives to fossil fuels. Using photosynthetic microorganisms to produce such alternatives is advantageous, since they can achieve direct conversion of carbon dioxide from the atmosphere into the desired product, using sunlight as the energy source. Squalene is a naturally occurring 30-carbon isoprenoid, which has commercial use in cosmetics and in vaccines. If it could be produced sustainably on a large scale, it could also be used instead of petroleum as a raw material for fuels and as feedstock for the chemical industry. The unicellular cyanobacterium Synechocystis PCC 6803 possesses a gene, slr2089, predicted to encode squalene hopene cyclase (Shc), an enzyme converting squalene into hopene, the substrate for forming hopanoids. Through inactivation of slr2089 (shc), we explored the possibility to produce squalene using cyanobacteria. The inactivation led to accumulation of squalene, to a level over 70 times higher than in wild type cells, reaching 0.67 mg OD750(-1) L(-1). We did not observe any significant growth deficiency in the Δshc strain compared to the wild type Synechocystis, even at high light conditions, suggesting that the observed squalene accumulation was not detrimental to growth, and that formation of hopene by Shc is not crucial for growth under normal conditions, nor for high-light stress tolerance. Effects of different light intensities and growth stages on squalene accumulation in the Δshc strain were investigated. We also identified a gene, sll0513, as a putative squalene synthase in Synechocystis, and verified its function by inactivation. In this work, we show that it is possible to use the cyanobacterium Synechocystis to generate squalene, a hydrocarbon of commercial interest and a potential biofuel. We also report the first identification of a squalene hopene cyclase, and the second identification of squalene synthase, in cyanobacteria.

Project description:BackgroundMetabolic engineering and synthetic biology of cyanobacteria offer a promising sustainable alternative approach for fossil-based ethylene production, by using sunlight via oxygenic photosynthesis, to convert carbon dioxide directly into ethylene. Towards this, both well-studied cyanobacteria, i.e., Synechocystis sp PCC 6803 and Synechococcus elongatus PCC 7942, have been engineered to produce ethylene by introducing the ethylene-forming enzyme (Efe) from Pseudomonas syringae pv. phaseolicola PK2 (the Kudzu strain), which catalyzes the conversion of the ubiquitous tricarboxylic acid cycle intermediate 2-oxoglutarate into ethylene.ResultsThis study focuses on Synechocystis sp PCC 6803 and shows stable ethylene production through the integration of a codon-optimized version of the efe gene under control of the Ptrc promoter and the core Shine-Dalgarno sequence (5'-AGGAGG-3') as the ribosome-binding site (RBS), at the slr0168 neutral site. We have increased ethylene production twofold by RBS screening and further investigated improving ethylene production from a single gene copy of efe, using multiple tandem promoters and by putting our best construct on an RSF1010-based broad-host-self-replicating plasmid, which has a higher copy number than the genome. Moreover, to raise the intracellular amounts of the key Efe substrate, 2-oxoglutarate, from which ethylene is formed, we constructed a glycogen-synthesis knockout mutant (ΔglgC) and introduced the ethylene biosynthetic pathway in it. Under nitrogen limiting conditions, the glycogen knockout strain has increased intracellular 2-oxoglutarate levels; however, surprisingly, ethylene production was lower in this strain than in the wild-type background.ConclusionMaking use of different RBS sequences, production of ethylene ranging over a 20-fold difference has been achieved. However, a further increase of production through multiple tandem promoters and a broad-host plasmid was not achieved speculating that the transcription strength and the gene copy number are not the limiting factors in our system.

Dataset Information

Characterization of a Highly pH Stable Chi-Class Glutathione S-Transferase from Synechocystis PCC 6803.

Publications

Characterization of a Highly pH Stable Chi-Class Glutathione S-Transferase from Synechocystis PCC 6803.

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