الفهرس 
Review of LiteratureOnly 14 pages are availabe for public view
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Abstract
Metagenomics is a powerful tool for studying the genomes of a wide range of unculturable microbes and investigating their potential as sources of novel enzymes. The deep-sea brines of the Red Sea are remote and unexplored environments characterized by high temperatures, low oxygen and elevated concentrations of salt and heavy metals. The metagenomic studies on the Red Sea brine pools started in March/April (2010) by the KAUST/WHOI/HCMR oceanographic expedition cruise, which aimed at revealing the microbial community in these hot pools. A metagenomic dataset was established for the microbial community that resides in the lower convective layer in Atlantis II deep, which represents the highest temperature and salinity region in the Red Sea and associated with metalliferous sediments of potential economic importance.
In completion of these metagenomics-based studies of the Red Sea brine pools, this study was conducted to isolate, identify and characterize thioredoxin reductase enzyme from ATII-LCL brine pool, as a new promising source of the enzyme that can be used in industrial or biotechnological applications.
I. Sequence analysis
Thioredoxin reductase isolated from the metagenomic DNA extracted from the microbial community at LCL of Atlantis II deep (ATII-TrxR) showed a sequence identity of 87% with TrxR of Cupriavidus metallidurans. The sequence was aligned to nr BLASTX and screened against the conserved domain database and InterPro Web interfaces. The results showed that the protein sequence belonged to pyridine nucleotide-disulphide oxidoreductase; class-II family and contains two distinctive domains (cyclic nucleotide-binding and FAD/NAD(P)-binding domains).
II. Multiple sequences alignment and phylogenetic tree construction
The ATII-TrxR protein sequence was aligned against 12 different sequences of TrxR including two classes of TrxR (low & high). Alignments showed that the ATII-TrxR is classified as a high molecular weight class of TrxR. The conserved FAD binding domain was identified in all sequences (GXGXXG). Also, NADPH binding motif was detected (GGGXXA). The Neighbour-Joining phylogenetic tree showed that the ATII-TrxR was closely related to the sequences of heavy metal resistant bacteria Cupriavidus metallidurans and Cupriavidus HMR-1.
III. Modeling of the three-dimensional structure of ATII-TrxR enzyme
Prediction of the three-dimensional structure of ATII-TrxR protein sequence revealed that the ATII-TrxR enzyme was made up of three domains, including FAD and NADPH binding domains, which contained the catalytic and the active sites of the enzyme. An additional domain representing Crp superfamily and its binding domain for cNMP were also identified at the N-terminal of the structure.
IV. Evaluation of the number of salt bridges and hydrogen bonds in ATII-TrxR
The number of salt bridges and H-bonds in ATII-TrxR were calculated and compared with those of the respective TrxR enzymes from normal (mouse type2) and harsh (Cupriavidus metallidurans and HMR-1) environments. It was found that the number of salt bridges in ATII-TrxR was 4 times higher than that in TrxR from the normal habitat, while it was closely related to that of the enzymes isolated from extreme environments. Also, the predicted number of H-bonds in ATII-TrxR was found to be 373, which was higher than that of TrxRs from the best hit bacteria; Cupriavidus metallidurans and Cupriavidus HMR-1 (353 & 351, respectively), as well as the mouse type 2 (340).
V. Physicochemical properties of ATII-TrxR protein sequence
The amino acids sequence of ATII-TrxR was analyzed and compared to that of bacterial TrxR from the first hit Cupriavidus metallidurans. ATII-TrxR sequence was found to contain a higher percentage of negatively and positively charged amino acid than TrxR sequence of Cupriavidus metallidurans by 0.87 and 0.865%, respectively. These results were confirmed by analyzing the frequency of substitutions of each amino acid in ATII-TrxR enzyme compared to the protein sequence of TrxR from Cupriavidus metallidurans. It was found that alanine, threonine, glutamic acid, aspartic acid, arginine and histidine were higher in ATII-TrxR than those present in Cupriavidus metallidurans, while glutamine and asparagine were lower.
VI. Expression and purification of ATII-TrxR.
ATII-TrxR gene was synthesized and optimized to allow optimal expression in E. coli. The gene was cloned into pET 28a (+) expression vector system (Novagen, USA) and its expressed protein was determined using SDS-PAGE. The purified protein had a molecular weight of 57.8 KDa. The purified fractions were then collected and dialyzed by ÄKTA purifier.
VII. Redox activity and kinetics parameters of ATII-TrxR
The catalytic activity of ATII-TrxR was estimated. The enzyme was able to reduce its substrate thioredoxin (isolated from the same environment at Atlantis II deep), with a km and Kcat values of 0.103 µM and 28.4 S-1, respectively. Also, the enzyme reduced the DTNB (model substrate for TrxR enzymes) with km and Kcat values of 98.32 µM and 5.63 S-1, respectively. Additionally, ATII-TrxR showed a high specificity towards NADPH as a cofactor recording kinetics values of 5.21 µM and 8.96 S-1 for the km and Kcat, respectively.
VIII. Halophilicity of ATII-TrxR enzyme
ATII-TrxR enzyme activity was found to be increased by increasing the concentration of NaCl, reaching its maximum activity of 27.71 µmol/min/mg at 3 M NaCl, then the activity was decreased slightly at 4 M reaching 23.58 µmol/min/mg.
IX. Thermal stability of ATII-TrxR enzyme
ATII-TrxR showed a remarkable thermal stability, where the enzyme retained 60% of its activity after incubation at 70°C for 10-min, whereas it was remarkably reduced attaining 39 % of its activity following incubation at 99°C for 10 min. Also, the thermal stability of ATII-TrxR was studied by incubating the enzyme at two temperatures (70 and 90 oC) at different time periods (0-60 min). ATII-TrxR enzyme retained 43 and 33% of its activity after incubation for 30 min at 70 and 90 oC, respectively. By increasing the duration of the thermal treatment, the enzyme started to lose most of its activity recording 20 and 13% of its maximum activity after incubation for 1 h at 70 and 90 oC, respectively.