Molecular basis of function and the unusual antioxidant activity of a cyanobacterial cysteine desulfurase

2017 ◽  
Vol 474 (14) ◽  
pp. 2435-2447 ◽  
Author(s):  
Manisha Banerjee ◽  
Dhiman Chakravarty ◽  
Anand Ballal
Keyword(s):  
Oxidative Stress ◽  
Catalytic Efficiency ◽  
Cysteine Desulfurase ◽  
Iron Sulfur Cluster ◽  
Utilization Factor ◽  
Reading Frame ◽  
Anabaena Pcc 7120 ◽  
Cyanobacterium Anabaena ◽  
Pcc 7120 ◽  
Cluster Biosynthesis

Cysteine desulfurases, which supply sulfur for iron–sulfur cluster biogenesis, are broadly distributed in all phyla including cyanobacteria, the progenitors of plant chloroplasts. The SUF (sulfur utilization factor) system is responsible for Fe–S cluster biosynthesis under stress. The suf operon from cyanobacterium Anabaena PCC 7120 showed the presence of a cysteine desulfurase, sufS (alr2495), but not the accessory sulfur-accepting protein (SufE). However, an open reading frame (alr3513) encoding a SufE-like protein (termed AsaE, Anabaena sulfur acceptor E) was found at a location distinct from the suf operon. The purified SufS protein existed as a pyridoxal 5' phosphate (PLP)-containing dimer with a relatively low desulfurase activity. Interestingly, in the presence of the AsaE protein, the catalytic efficiency of this reaction increased 10-fold. In particular, for sulfur mobilization, the AsaE protein partnered only SufS and not other cysteine desulfurases from Anabaena. The SufS protein was found to physically interact with the AsaE protein, demonstrating that AsaE was indeed the missing partner of Anabaena SufS. The conserved cysteine of the SufS or the AsaE protein was essential for activity but not for their physical association. Curiously, overexpression of the SufS protein in Anabaena caused reduced formation of reactive oxygen species on exposure to hydrogen peroxide (H2O2), resulting in superior oxidative stress tolerance to the oxidizing agent when compared with the wild-type strain. Overall, the results highlight the functional interaction between the two proteins that mediate sulfur mobilization, in the cyanobacterial SUF pathway, and further reveal that overexpression of SufS can protect cyanobacteria from oxidative stress.

scholarly journals Molecular Characterization of a Novel Peroxidase from the Cyanobacterium Anabaena sp. Strain PCC 7120

2009 ◽  
Vol 75 (23) ◽  
pp. 7509-7518 ◽  
Author(s):  
Henry Joseph Oduor Ogola ◽  
Takaaki Kamiike ◽  
Naoya Hashimoto ◽  
Hiroyuki Ashida ◽  
Takahiro Ishikawa ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Active Form ◽  
Catalytic Efficiency ◽  
Synthetic Dyes ◽  
Reading Frame ◽  
Anthraquinone Dyes ◽  
Aromatic Substrates ◽  
Cyanobacterium Anabaena ◽  
Optimum Reaction ◽  
Pcc 7120

ABSTRACT The open reading frame alr1585 of Anabaena sp. strain PCC 7120 encodes a heme-dependent peroxidase (Anabaena peroxidase [AnaPX]) belonging to the novel DyP-type peroxidase family (EC 1.11.1.X). We cloned and heterologously expressed the active form of the enzyme in Escherichia coli. The purified enzyme was a 53-kDa tetrameric protein with a pI of 3.68, a low pH optima (pH 4.0), and an optimum reaction temperature of 35°C. Biochemical characterization revealed an iron protoporphyrin-containing heme peroxidase with a broad specificity for aromatic substrates such as guaiacol, 4-aminoantipyrine and pyrogallol. The enzyme efficiently catalyzed the decolorization of anthraquinone dyes like Reactive Blue 5, Reactive Blue 4, Reactive Blue 114, Reactive Blue 119, and Acid Blue 45 with decolorization rates of 262, 167, 491, 401, and 256 μM·min−1, respectively. The apparent Km and k cat/Km values for Reactive Blue 5 were 3.6 μM and 1.2 × 107 M−1 s−1, respectively, while the apparent Km and k cat/Km values for H2O2 were 5.8 μM and 6.6 × 106 M−1 s−1, respectively. In contrast, the decolorization activity of AnaPX toward azo dyes was relatively low but was significantly enhanced 2- to ∼50-fold in the presence of the natural redox mediator syringaldehyde. The specificity and catalytic efficiency for hydrogen donors and synthetic dyes show the potential application of AnaPX as a useful alternative of horseradish peroxidase or fungal DyPs. To our knowledge, this study represents the only extensive report in which a bacterial DyP has been tested in the biotransformation of synthetic dyes.

A novel glutaredoxin domain-containing peroxiredoxin ‘All1541’ protects the N2-fixing cyanobacterium Anabaena PCC 7120 from oxidative stress

2012 ◽  
Vol 442 (3) ◽  
pp. 671-680 ◽  
Author(s):  
Manisha Banerjee ◽  
Anand Ballal ◽  
Shree K. Apte
Keyword(s):  
Oxidative Stress ◽  
Peroxidase Activity ◽  
Bioinformatic Analysis ◽  
Open Reading Frames ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Anabaena Pcc 7120 ◽  
Cyanobacterium Anabaena ◽  
Pcc 7120

Prxs (peroxiredoxins) are ubiquitous thiol-based peroxidases that detoxify toxic peroxides. The Anabaena PCC 7120 genome harbours seven genes/ORFs (open reading frames) which have homology with Prxs. One of these (all1541) was identified to encode a novel Grx (glutaredoxin) domain-containing Prx by bioinformatic analysis. A recombinant N-terminal histidine-tagged All1541 protein was overexpressed in Escherichia coli and purified. Analysis with the protein alkylating agent AMS (4-acetamido-4′-maleimidyl-stilbene-2,2′-disulfonate) showed All1541 to form an intra-molecular disulfide bond. The All1541 protein used glutathione (GSH) more efficiently than Trx (thioredoxin) to detoxify H2O2. Deletion of the Grx domain from All1541 resulted in loss of GSH-dependent peroxidase activity. Employing site-directed mutagenesis, the cysteine residues at positions 50 and 75 were identified as peroxidatic and resolving cysteine residues respectively, whereas both the cysteine residues within the Grx domain (positions 181 and 184) were shown to be essential for GSH-dependent peroxidase activity. On the basis of these data, a reaction mechanism has been proposed for All1541. In vitro All1541 protein protected plasmid DNA from oxidative damage. In Anabaena PCC 7120, all1541 was transcriptionally activated under oxidative stress. Recombinant Anabaena PCC 7120 strain overexpressing All1541 protein showed superior oxidative stress tolerance to H2O2 as compared with the wild-type strain. The results suggest that the glutathione-dependent peroxidase All1541 plays an important role in protecting Anabaena from oxidative stress.

scholarly journals Iron–sulfur cluster biosynthesis

2008 ◽  
Vol 36 (6) ◽  
pp. 1112-1119 ◽  
Author(s):  
Sibali Bandyopadhyay ◽  
Kala Chandramouli ◽  
Michael K. Johnson
Keyword(s):  
Scaffold Proteins ◽  
Cluster Assembly ◽  
Cysteine Desulfurase ◽  
Iron Sulfur Cluster ◽  
Iron Sulfur ◽  
Eukaryotic Organisms ◽  
Almost All ◽  
Cluster Biosynthesis

Iron–sulfur (Fe–S) clusters are present in more than 200 different types of enzymes or proteins and constitute one of the most ancient, ubiquitous and structurally diverse classes of biological prosthetic groups. Hence the process of Fe–S cluster biosynthesis is essential to almost all forms of life and is remarkably conserved in prokaryotic and eukaryotic organisms. Three distinct types of Fe–S cluster assembly machinery have been established in bacteria, termed the NIF, ISC and SUF systems, and, in each case, the overall mechanism involves cysteine desulfurase-mediated assembly of transient clusters on scaffold proteins and subsequent transfer of pre-formed clusters to apo proteins. A molecular level understanding of the complex processes of Fe–S cluster assembly and transfer is now beginning to emerge from the combination of in vivo and in vitro approaches. The present review highlights recent developments in understanding the mechanism of Fe–S cluster assembly and transfer involving the ubiquitous U-type scaffold proteins and the potential roles of accessory proteins such as Nfu proteins and monothiol glutaredoxins in the assembly, storage or transfer of Fe–S clusters.

Sodium regulation of GAF domain function

2007 ◽  
Vol 35 (5) ◽  
pp. 1032-1034 ◽  
Author(s):  
M.J. Cann
Keyword(s):  
Cyclic Nucleotides ◽  
Protein Domain ◽  
Genetic Ablation ◽  
Anabaena Pcc 7120 ◽  
Cyanobacterium Anabaena ◽  
Signal Changes ◽  
Sodium Regulation ◽  
Pcc 7120 ◽  
Camp And Cgmp

Cyclic nucleotide PDEs (phosphodiesterases) regulate cellular levels of cAMP and cGMP by controlling the rate of degradation. Several mammalian PDE isoforms possess N-terminal GAF (found in cGMP PDEs, Anabaena adenylate cyclases and Escherichia coli FhlA; where FhlA is formate hydrogen lyase transcriptional activator) domains that bind cyclic nucleotides. Similarly, the CyaB1 and CyaB2 ACs (adenylate cyclases) of the cyanobacterium Anabaena PCC 7120 bind cAMP through one (CyaB1) or two (CyaB2) N-terminal GAF domains and mediate autoregulation of the AC domain. Sodium inhibits the activity of CyaB1, CyaB2 and mammalian PDE2A in vitro through modulation of GAF domain function. Furthermore, genetic ablation of cyaB1 and cyaB2 gives rise to Anabaena strains defective in homoeostasis at limiting sodium. Sodium regulation of GAF domain function has therefore been conserved since the eukaryotic/prokaryotic divergence. The GAF domain is the first identified protein domain to directly sense and signal changes in environmental sodium.

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