Table 1.
InterPro, CDD and nr Blastx search results for the open reading frame (ORF) selected in this study.
Fig 1.
Multiple sequence alignment of ATII-TrxR with thioredoxin reductases from different species.
The alignments were carried out using ClustalX version 2.1. Conserved amino acids are coloured. As shown in the figure, the FAD-binding domain is conserved in all the thioredoxin reductases and identifiable by the GXGXXG sequence. Additionally, the NADPH-binding motif was detected in all the sequences (GGGXXA). ATII-TrxR was aligned against 16 different TrxRs from different species and environments: WP_072867591.1 (Desulfotomaculum thermosubterraneum), WP_013274997.1 (Thermosediminibacter oceani), WP_068550046.1 (Thermosulfidibacter takaii), SDK15280.1 (Jeotgalicoccus halophilus), WP_046789418.1 (Salinicoccus halodurans), WP_072709726.1 (Salinicoccus alkaliphilus), WP_026799979.1 (Pontibacillus halophilus), WP_003349406.1 (Bacillus methanolicus), WP_015009427.1 (Amphibacillus xylanus), WP_013451428.1 (Calditerrivibrio nitroreducens), WP_008942829.1| (Oceanibaculum indicum), ADT74500.1 (Escherichia coli W), WP_011516654.1 (Cupriavidus metallidurans), WP_008651587.1 (Cupriavidus sp. HMR-1), WP_075466512.1 (Ralstonia solanacearum) and WP_053838757.1 (Xanthomonas translucens).
Fig 2.
Phylogenetic analysis of ATII-TrxR.
The evolutionary history of ATII-TrxR with thioredoxin reductases from different species was inferred using the neighbour-joining method. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The analysis involved 37 amino acid sequences. Evolutionary analyses were conducted in MEGA7. As shown in the figure, the phylogenetic tree of the thioredoxin reductase protein sequence extracted from the LCL of Atlantis II brine pools of the Red Sea revealed that the ATII-TrxR sequence was closely related to the sequences from the heavy metal-resistant bacteria Cupriavidus metallidurans and Cupriavidus sp. HMR-1.
Fig 3.
Predicted three-dimensional structural model of ATII-TrxR.
The structure is composed of an N-terminal domain (blue) and a C-terminal domain (red). Both the FAD- and NADPH-binding domains are illustrated in the figure, and the additional domain representing the Crp superfamily and its binding domain for cNMP are shown. Also, the cysteine residues involved in the redox centres (Cys206-Cys223) and (Cys376-Cys467) are illustrated.
Fig 4.
Identification of functional regions in ATII-TrxR enzyme sequence by the ConSurf server.
The ConSurf server, based on multiple sequence alignments, predicted that almost all of the glutamic acid (E), arginine (R) and glycine (G) residues are functional residues that are highly conserved and exposed to the outside of the ATII-TrxR protein structure.
Table 2.
Predicted number of salt bridges and H-bonds in ATII-TrxR enzyme and the corresponding enzyme templates.
Fig 5.
a) Distribution percentages of different classes of amino acids in ATII-TrxR relative to that of Cupriavidus metallidurans. b) Pattern of amino acid substitutions in ATII-TrxR relative to those of Cupriavidus metallidurans. a) The amino acid compositions of TrxR from ATII-LCL and Cupriavidus metallidurans were compared, and the histograms show the net change in the number of each of the listed amino acids in the ATII-LCL enzyme. b) Frequency of substitutions in ATII-LCL plotted against the corresponding residue in the Cupriavidus metallidurans TrxR enzyme.
Fig 6.
SDS-PAGE of the recombinant ATII-TrxR protein.
Aliquots from the HisTag purification process of the ATII-TrxR protein from total bacterial proteins were analysed by 10% SDS-PAGE as described in the experimental procedures. M: molecular weight marker; lane 1: induced sample along with all bacterial proteins; lanes 2 & 3: bacterial flow-through-1 & flow-through-2; lanes 4–9: different fractions of eluted and purified ATII-TrxR protein with a size of 57.8 kDa.
Table 3.
Kinetic parameters of ATII-TrxR with different substrates.
Fig 7.
Characterization of the ATII-TrxR enzyme.
a) Redox activity of the ATII-TrxR enzyme at different molar concentrations of NaCl (halophilicity). b) Effect of increasing temperature on ATII-TrxR enzyme activity. The figure shows that the enzyme is thermophilic with an optimum temperature of 65°C. c) Residual activity of ATII-TrxR at different temperatures in the absence and presence of 2 M NaCl. d) Thermal stability of ATII-TrxR as a function of time.