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• W1969384259 abstract "The open reading frame of TTHA0613 from Thermus thermophilus HB8, consisting of 94 amino acid residues, was annotated as a hypothetical protein, and its function was not inferred. A PSI-BLAST search,1 using the amino acid sequence of TTHA0613 as the query, identified a total of 10 homologues with E-values below 0.005. In the Clusters of Orthologous Groups of proteins2 database, two of these homologues belong to COG0607: rhodanese-related sulfurtransferases. Rhodaneses catalyze the transfer of the sulfane sulfur from thiosulfate to cyanide, forming thiocyanate and sulfite in vitro. The 3-mercaptopyruvate sulfurtransferases (MST), which share strong sequence similarity with the rhodaneses, catalyze the same sulfane sulfur transfer reaction, using 3-mercaptopyruvate as a sulfur donor in vitro.3 Rhodanese homology domains are conserved in all three kingdoms. Their in vivo functions have been seriously debated. The ∼110-amino acid rhodanese homology domains exist as single domain proteins, as tandem repeats with a C-terminal domain hosting the properly structured active-site Cys residue, and as members of multidomain proteins. The rhodanese domain fold was first observed in the crystal structure of bovine mitochondrial rhodanese (Rhobov).4 Subsequently, that of Azotobacter vinelandii rhodanese (RhoA) was determined.5 Each structure consists of two rhodanese domain folds in the N- and C-terminal halves of the polypeptide. On the other hand, GlpE from Escherichia coli, which displays rhodanese activity in vitro,6 is composed of a single rhodanese domain fold.7 Rhodanese homology domains were also observed in the crystal structures of the human Cdc25a8 and Cdc25b9 phosphatases. Several genes encoding distinct rhodanese homology domains exist in the same genomes. This fact suggests that the various rhodanese homology domains might have diverse biological functions.10 Here we report the crystal structure of TTHA0613 from Thermus thermophilus HB8 at 2.0 Å resolution, and discuss our sequence and structural comparisons with its homologues. The open reading frame of TTHA0613 from T. thermophilus HB8 was cloned into the pET11a expression vector (Novagen). SeMet-substituted proteins were produced in E. coli Rosetta834 (DE3). The cell lysate was heated at 70°C for 15 min, and the proteins were purified by a series of HiTrap Q HP, Resource Q, and Superdex 75 HR 10/30 (Amersham Biosciences) column chromatography steps. The purified protein was concentrated to 12.7 mg/mL using an Amicon Ultra 4 filter (Millipore). The crystals were obtained by the microbatch method at 20°C. The precipitant solution was 100 mM MES-NaOH (pH 6.0–6.3) containing 35–40% polyethylene glycol 4000. The crystal was cryo-cooled to −173°C after soaking in a cryoprotectant solution [95% (v/v) Paraton-N and 5% (v/v) glycerol]. X-ray diffraction data of the SeMet-derivative crystal were obtained on beamline BL26B1 at the SPring-8 (Harima, Japan), using a Jupiter210 CCD detector (RIGAKU/MSC). Diffraction data were processed with the HKL200011 program suite. The crystal structure was solved by the multiwavelength anomalous dispersion (MAD)12 method, using the programs SOLVE13 and RESOLVE.14 The refinement was carried out using the program CNS.15 The final model comprises 173 amino acid residues and 132 water molecules in the asymmetric unit. Residues 1, 26–27, and 91–94 of molecule A, and residues 26–28 and 90–94 of molecule B were not visible, due to disorder, and were excluded from the final model. Data collection, MAD phasing, and refinement statistics are summarized in Table I. We have determined the crystal structure of TTHA0613 from T. thermophilus HB8 at 2.0 Å resolution. The crystal contains two molecules, A and B, per asymmetric unit [Fig. 1(A)]. The root mean square deviations of all of the atoms between molecule A and B are 0.65 Å. The monomeric structure consists of a five-stranded parallel β-sheet, surrounded by three α-helices [Fig. 1(B)]. As expected from the PSI-BLAST search results, the structure adopts a single rhodanese domain fold. A DALI16 search revealed that TTHA0613 shares high structural similarity to rhodanese homology domains. The Z-scores are 10.4 for GlpE from E. coli, 9.9 for the catalytic domain of RhoA, 9.6 for the catalytic domain of Rhobov, 8.7 for MST from Leishmania major,17 8.3 for Sud and 7.5 for Cdc25a. TTHA0613 tends to be structurally more similar to the sulfurtransferases than the phosphatase. A structure-based sequence alignment of TTHA0613 and the rhodanese homology domains is shown in Figure 1(C). The putative active-site loop motif of TTHA0613 (CEKGLL) lacks similarity to the other catalytic rhodanese homology domains with structures that have already been determined [Fig. 1(C)]. However, the conformation of the putative active-site loop closely matches those of the other rhodaneses. The Cα–Cα RMSDs calculated over the six residues of the active-site loop yielded the small values of 0.23 Å for GlpE, 0.12 Å for RhoA, 0.22 Å for Rhobov, 0.15 Å for MST from L. major, and 0.68 Å for Cdc25a. The conformation of the putative active site is also similar to those of the sulfurtransferases. A sequence comparison between TTHA0613 and its six closest homologues, each with a single rhodanese homology domain, is shown in Figure 1(D). The high sequence homologies of β4 and α3 are probably necessary to form the proper active-site loop conformation. Furthermore, high sequence homology exists in the N-terminal β1–α1–β2 region. This region might participate in interactions with other proteins that bind to the in vivo substrate. (A). Crystal packing of the molecules in the asymmetric unit. Molecules A and B are colored pink and blue, respectively. (B) Ribbon representation of the TTHA0613 monomer (stereo view). The α-helices and β-strands are colored red and green, respectively. The figure was generated with the graphics program CueMol (http://cuemol.sourceforge.jp/en/). (C) Structure-based sequence alignment of TTHA0613 and rhodanese homology domains. (D) Sequence comparison between TTHA0613 and its six closest homologs, which each have a single rhodanese homology domain. The figure was constructed by PSI-BLAST1 followed by CLUSTALW.18 Ll, Lactococcus lactis subsp; Aa, Arthrobacter aurescens; Tt, Thermus thermophilus; Lp, Legionella pneumophila subs; Ss, Streptococcus suis; Ps, Pseudomonas syringae. Strictly conserved and similar residues are represented within a red box and by a red letter, respectively. The figure was generated with ESpript 2.0.19 We thank M. Yamamoto for data collection at BL26B1 of SPring-8. This work was supported by the RIKEN Structural Genomics/Proteomics Initiative (RSGI), the National Project on Protein Structural and Functional Analysis, Ministry of Education, Culture, Sports, Science and Technology of Japan." @default.
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• W1969384259 date "2006-05-05" @default.
• W1969384259 modified "2023-10-17" @default.
• W1969384259 title "Crystal structure of the single‐domain rhodanese homologue TTHA0613 from <i>Thermus thermophilus</i> HB8" @default.
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• W1969384259 doi "https://doi.org/10.1002/prot.20937" @default.
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