Amino-acid sequence of a tetrameric, manganese superoxide dismutase from Thermus thermophilus HB8

The amino-acid sequence of a tetrameric manganese superoxide dismutase from Thermus thermophilus HB8 has been determined. The protein was cleaved with cyanogen bromide (BrCN) into four peptides and their alignment was deduced through the fragment of partial cleavage with BrCN and the peptides were produced by cleavage of the protein with o-iodosobenzoic acid. Most of the peptides were sequenced by solid phase Edman degradation. Some of the peptides were sequenced by the Edman dansyl method after sub-fragmentation by proteinase digestion. The amino-acid sequence consists of 203 residues corresponding to a subunit molecular weight of 23,144.     

Magnetization of manganese superoxide dismutase from Thermus thermophilus.

The ground state magnetic properties of manganese superoxide dismutase from Thermus thermophilus in its native and reduced forms have been determined using saturation magnetization data. Parallel EPR measurements were used to verify that commonly encountered paramagnetic impurities were at low concentration relative to the metalloprotein. The native enzyme contains high spin Mn(III) (S = 2) with D = +2.44(5) cm-1 and E/D = 0. The reduced enzyme contains high spin Mn(II) (S = 5/2) with D = +0.50(5) cm-1 and E/D = 0.027. These results are in keeping with the suggestions of several previous groups of workers concerning the permissible oxidation and spin states of the manganese, but the zero field splitting parameters are unlike those of known manganese model compounds. In addition, the extinction coefficient for the visible region absorption maximum of the native enzyme and the corresponding difference extinction coefficient (native minus reduced) have been measured using saturation magnetization data to quantitate Mn(III) present. The result, epsilon 480 = 950(80) M-1 cm-1 (delta epsilon 480 = 740(60) M-1 cm-1) agrees with the previously reported value of epsilon 480 = 910 M-1 cm-1 found by total manganese determination (Sato, S. and Nakazawa, K. (1978) J. Biochem. 83, 1165-1171). The wide variation in the reported visible region extinction coefficients of manganese superoxide dismutases from different sources is discussed.     

Three-dimensional structure of phenylalanyl-transfer RNA synthetase from Thermus thermophilus HB8 at 0.6-nm resolution.

The three-dimensional structure of the heterodimeric alpha 2 beta 2 enzyme phenylalanyl-tRNA synthetase from Thermus thermophilus HB8 has been determined by X-ray crystallography, using the multiple-isomorphous-replacement method at 0.6 nm resolution. Trigonal crystals of space group P3(2)21 have cell dimensions a = b = 17.6 nm and c = 14.2 nm. Assuming one heterodimeric molecule/asymmetric unit, the ratio of unit cell volume/molecular mass was V = 0.00244 nm3/Da, which is in the middle of the range normally observed. However, after a rotation-function calculation and measurement of the density of the native crystals, we postulate the existence of only the alpha beta dimer in the asymmetric units. This implies 73% solvent content in the unit cell. Three heavy-atom derivatives [K2PtCl4, KAu(CN)2 and Hg(CH3COO)2] and the solvent-flattening procedure were used for electron-density-map calculations. This map confirmed our hypothesis and revealed a remarkably large space filled by solvent, with alpha beta dimer only in the asymmetric unit. The phenylalanyl-tRNA synthetase from T. thermophilus molecule has a 'quasi-linear' subunit organization. As can be concluded at this level of resolution, there is no contact between small alpha subunits in the functional heterodimer.     

Manganese superoxide dismutase from Thermus thermophilus. A structural model refined at 1.8 A resolution

The structure of Mn(III) superoxide dismutase (Mn(III)SOD) from Thermus thermophilus, a tetramer of chains 203 residues in length, has been refined by restrained least-squares methods. The R-factor [formula: see text] for the 54,056 unique reflections measured between 10.0 and 1.8 A (96% of all possible reflections) is 0.176 for a model comprising the protein dimer and 180 bound solvents, the asymmetric unit of the P4(1)2(1)2 cell. The monomer chain forms two domains as determined by distance plots: the N-terminal domain is dominated by two long antiparallel helices (residues 21 to 45 and 69 to 89) and the C-terminal domain (residues 100 to 203) is an alpha + beta structure including a three-stranded sheet. Features that may be important for the folding and function of this MnSOD include: (1) a cis-proline in a turn preceding the first long helix; (2) a residue inserted at position 30 that distorts the helix near the first Mn ligand; and (3) the locations of glycine and proline residues in the domain connector (residues 92 to 99) and in the vicinity of the short cross connection (residues 150 to 159) that links two strands of the beta-sheet. Domain-domain contacts include salt bridges between arginine residues and acidic side chains, an extensive hydrophobic interface, and at least ten hydrogen-bonded interactions. The tetramer possesses 222 symmetry but is held together by only two types of interfaces. The dimer interface at the non-crystallographic dyad is extensive (1000 A2 buried surface/monomer) and incorporates 17 trapped or structural solvents. The dimer interface at the crystallographic dyad buries fewer residues (750 A2/monomer) and resembles a snap fastener in which a type I turn thrusts into a hydrophobic basket formed by a ring of helices in the opposing chain. Each of the metal sites is fully occupied, with the Mn(III) five-co-ordinate in trigonal bipyramidal geometry. One of the axial ligands is solvent; the four protein ligands are His28, His83, Asp166 and His170. Surrounding the metal-ligand cluster is a shell of predominantly hydrophobic residues from both chains of the asymmetric unit (Phe86A, Trp87A, Trp132A, Trp168A, Tyr183A, Tyr172B, Tyr173B), and both chains collaborate in the formation of a solvent-lined channel that terminates at Tyr36 and His32 near the metal ion and is presumed to be the path by which substrate or other inner-sphere ligands reach the metal.(ABSTRACT TRUNCATED AT 400 WORDS)     

Crystal structure of a predicted phosphoribosyltransferase (TT1426) from Thermus thermophilus HB8 at 2.01 A resolution

TT1426, from Thermus thermophilus HB8, is a conserved hypothetical protein with a predicted phosphoribosyltransferase (PRTase) domain, as revealed by a Pfam database search. The 2.01 A crystal structure of TT1426 has been determined by the multiwavelength anomalous dispersion (MAD) method. TT1426 comprises a core domain consisting of a central five-stranded beta sheet surrounded by four alpha-helices, and a subdomain in the C terminus. The core domain structure resembles those of the type I PRTase family proteins, although a significant structural difference exists in an inserted 43-residue region. The C-terminal subdomain corresponds to the "hood," which contains a substrate-binding site in the type I PRTases. The hood structure of TT1426 differs from those of the other type I PRTases, suggesting the possibility that TT1426 binds an unknown substrate. The structure-based sequence alignment provides clues about the amino acid residues involved in catalysis and substrate binding.     

Crystal structure of ribosomal protein L27 from Thermus thermophilus HB8

Ribosomal protein L27 is located near the peptidyltransferase center at the interface of ribosomal subunits, and is important for ribosomal assembly and function. We report the crystal structure of ribosomal protein L27 from Thermus thermophilus HB8, which was determined by the multiwavelength anomalous dispersion method and refined to an R-factor of 19.7% (R(free) = 23.6%) at 2.8 A resolution. The overall fold is an all beta-sheet hybrid. It consists of two sets of four-stranded beta-sheets formed around a well-defined hydrophobic core, with a highly positive charge on the protein surface. The structure of ribosomal protein L27 from T. thermophilus HB8 in the RNA-free form is investigated, and its functional roles in the ribosomal subunit are discussed.     

Crystal structure of monofunctional histidinol phosphate phosphatase from Thermus thermophilus HB8

Monofunctional histidinol phosphate phosphatase from Thermus thermophilus HB8, which catalyzes the dephosphorylation of l-histidinol phosphate, belongs to the PHP family, together with the PHP domain of bacterial DNA polymerase III and family X DNA polymerase. We have determined the structures of the complex with a sulfate ion, the complex with a phosphate ion, and the unliganded form at 1.6, 2.1, and 1.8 A resolution, respectively. The enzyme exists as a tetramer, and the subunit consists of a distorted (betaalpha)7 barrel with one linker and one C-terminal tail. Three metal sites located on the C-terminal side of the barrel are occupied by Fe1, Fe2, and Zn ions, respectively, forming a trinuclear metal center liganded by seven histidines, one aspartate, one glutamate, and one hydroxide with two Fe ions bridged by the hydroxide. In the complexes, the sulfate or phosphate ion is coordinated to three metal ions, resulting in octahedral, trigonal bipyramidal, and tetrahedral geometries around the Fe1, Fe2, and Zn ions, respectively. The ligand residues are derived from the four motifs that characterize the PHP family and from two motifs conserved in histidinol phosphate phosphatases. The (betaalpha)7 barrel and the metal cluster are closely related in nature and architecture to the (betaalpha)8 barrel and the mononuclear or dinuclear metal center in the amidohydrolase superfamily, respectively. The coordination behavior of the phosphate ion toward the metal center supports the mechanism in which the bridging hydroxide makes a direct attack on the substrate phosphate tridentately bound to the two Fe ions and Zn ion to hydrolyze the phosphoester bond.     

Crystal structure of the GTP-binding protein Obg from Thermus thermophilus HB8

Obg comprises a unique family of high-molecular mass GTPases conserved from bacteria to eukaryotes. Bacterial Obg is essential for cellular growth, sporulation, and differentiation. Here, we report the crystal structure of the full-length form of Obg from Thermus thermophilus HB8 at 2.07 A resolution, in the nucleotide-free state. It reveals a three-domain arrangement, composed of the N-terminal domain, the guanine nucleotide-binding domain (G domain), and the C-terminal domain. The N-terminal and G domains have the Obg fold and the Ras-like fold, respectively. These global folds are similar to those of the recently published structure of the C-terminal domain-truncated form of Obg from Bacillus subtilis. On the other hand, the C-terminal domain of Obg was found to have a novel fold (the OCT fold). A comparison of the T.thermophilus and B.subtilis nucleotide-free Obg structures revealed significant conformational changes in the switch-I and switch-II regions of the G domain. Notably, the N-terminal domain is rotated drastically, by almost 180 degrees, around the G domain axis. In the T.thermophilus Obg crystal, the nucleotide-binding site of the G domain interacts with the C-terminal domain of the adjacent molecule. These data suggest a possible domain rearrangement of Obg, and a potential role of the C-terminal domain in the regulation of the nucleotide-binding state.     

Properties and crystal structure of methylenetetrahydrofolate reductase from Thermus thermophilus HB8

Background

Methylenetetrahydrofolate reductase (MTHFR) is one of the enzymes involved in homocysteine metabolism. Despite considerable genetic and clinical attention, the reaction mechanism and regulation of this enzyme are not fully understood because of difficult production and poor stability. While recombinant enzymes from thermophilic organisms are often stable and easy to prepare, properties of thermostable MTHFRs have not yet been reported.

Methodology/Principal Findings

MTHFR from Thermus thermophilus HB8, a homologue of Escherichia coli MetF, has been expressed in E. coli and purified. The purified MTHFR was chiefly obtained as a heterodimer of apo- and holo-subunits, that is, one flavin adenine dinucleotide (FAD) prosthetic group bound per dimer. The crystal structure of the holo-subunit was quite similar to the β₈α₈ barrel of E. coli MTHFR, while that of the apo-subunit was a previously unobserved closed form. In addition, the intersubunit interface of the dimer in the crystals was different from any of the subunit interfaces of the tetramer of E. coli MTHFR. Free FAD could be incorporated into the apo-subunit of the purified Thermus enzyme after purification, forming a homodimer of holo-subunits. Comparison of the crystal structures of the heterodimer and the homodimer revealed different intersubunit interfaces, indicating a large conformational change upon FAD binding. Most of the biochemical properties of the heterodimer and the homodimer were the same, except that the homodimer showed ≈50% activity per FAD-bound subunit in folate-dependent reactions.

Conclusions/Significance

The different intersubunit interfaces and rearrangement of subunits of Thermus MTHFR may be related to human enzyme properties, such as the allosteric regulation by S-adenosylmethionine and the enhanced instability of the Ala222Val mutant upon loss of FAD. Whereas E. coli MTHFR was the only structural model for human MTHFR to date, our findings suggest that Thermus MTHFR will be another useful model for this important enzyme.     

Crystal structure of atypical cytoplasmic ABC-ATPase SufC from Thermus thermophilus HB8

SufC, a cytoplasmic ABC-ATPase, is one of the most conserved Suf proteins. SufC forms a stable complex with SufB and SufD, and the SufBCD complex interacts with other Suf proteins in the Fe-S cluster assembly. We have determined the crystal structure of SufC from Thermus thermophilus HB8 in nucleotide-free and ADP-Mg-bound states at 1.7A and 1.9A resolution, respectively. The overall architecture of the SufC structure is similar to other ABC ATPases structures, but there are several specific motifs in SufC. Three residues following the end of the Walker B motif form a novel 3(10) helix which is not observed in other ABC ATPases. Due to the novel 3(10) helix, a conserved glutamate residue involved in ATP hydrolysis is flipped out. Although this unusual conformation is unfavorable for ATP hydrolysis, salt-bridges formed by conserved residues and a strong hydrogen-bonding network around the novel 3(10) helix suggest that the novel 3(10) helix of SufC is a rigid conserved motif. Compared to other ABC-ATPase structures, a significant displacement occurs at a linker region between the ABC alpha/beta domain and the alpha-helical domain. The linker conformation is stabilized by a hydrophobic interaction between conserved residues around the Q loop. The molecular surfaces of SufC and the C-terminal helices of SufD (PDB code: 1VH4) suggest that the unusual linker conformation conserved among SufC proteins is probably suitable for interacting with SufB and SufD.     

Crystal structure of a novel polyisoprenoid-binding protein from Thermus thermophilus HB8

The isoprenoid quinones exist widely among prokaryotes and eukaryotes. They play essential roles in respiratory electron transport and in controlling oxidative stress and gene regulation. In the isoprenoid quinone biosynthetic pathway, polyprenyl pyrophosphates are used as isoprenoid side-chain precursors. Here we report the crystal structure of a novel polyprenyl pyrophosphate binding protein, TT1927b, from Thermus thermophilus HB8, complexed with its ligand. This protein belongs to the YceI-like family in the Pfam database, and its sequence homologs are present in a broad range of bacteria and archaea. The structure consists of an extended, eight-stranded, antiparallel beta-barrel. In the hydrophobic pore of the barrel, the protein binds the polyisoprenoid chain by hydrophobic interactions. Its overall structure resembles the lipocalin fold, but there is no sequence homology between TT1927b and the lipocalin family of proteins.     

Solution structure of ribosomal protein L16 from Thermus thermophilus HB8

Ribosomal protein L16 is an essential component of the bacterial ribosome. It organizes the architecture of aminoacyl tRNA binding site in the ribosome 50S subunit. The three-dimensional structure of L16 from Thermus thermophilus HB8 was determined by NMR. In solution, L16 forms an alpha+beta sandwich structure combined with two additional beta sheets located at the loop regions connecting the two layers. The terminal regions and a central loop region did not show any specific secondary structure. The structured part of L16 could be superimposed well on the C(alpha) model of L16 determined in the crystal structure of the ribosome 50S subunit. By overlaying the L16 solution structure onto the coordinates of the ribosome crystal structure, we constructed the combined model that represents the ribosome-bound state of L16 in the detailed structure. The model showed that L16 possesses residues in contact with helices 38, 39, 42, 43 and 89 of 23S rRNA and helix 4 of 5S rRNA. This suggests its broad effect on the ribosome architecture. Comparison of L16 with the L10e protein, which is the archaeal counterpart, showed that they share a common fold, but differ in some regions of functional importance, especially in the N-terminal region. All known mutation sites in L16 that confer resistance to avilamycin and evernimicin were positioned so that their side-chains were exposed to solvent in the internal cavity of the ribosome. This suggests the direct participation of L16 as a part of the binding site for antibiotics.     

The 2.1-A resolution structure of iron superoxide dismutase from Pseudomonas ovalis

The 2.1-A resolution crystal structure of native uncomplexed iron superoxide dismutase (EC 1.15.1.1) from Pseudomonas ovalis was solved and refined to a final R factor of 24%. The dimeric structure contains one catalytic iron center per monomer with an asymmetric trigonal-bipyramidal coordination of protein ligands to the metal. Each monomer contains two domains, with the trigonal ligands (histidines 74 and 160; aspartate 156) contributed by the large domain and stabilized by an extended hydrogen-bonded network, including residues from opposing monomers. The axial ligand (histidine 26) is found on the small domain and does not participate extensively in the stabilizing H-bond network. The open axial coordination position of the iron is devoid of bound water molecules or anions. The metal is located 0.5 A out of the plane of the trigonal ligands toward histidine 26, providing a slightly skewed coordination away from the iron binding site. The molecule contains a glutamine residue in the active site which is conserved between all iron enzymes sequenced to data but which is conserved among all manganese SODs at a separate position in the sequence. This residue shows the same structural interactions in both cases, implying that iron and manganese SODs are second-site revertants of one another.     

Structure of peptidoglycan from Thermus thermophilus HB8.

The composition and structure of peptidoglycan (murein) extracted from the extreme thermophilic eubacterium Thermus thermophilus HB8 are presented. The structure of 29 muropeptides, accounting for more than 85% of total murein, is reported. The basic monomeric subunit consists of N-acetylglucosamine-N-acetylmuramic acid-L-Ala-D-Glu-L-Orn-D-Ala-D-Ala, acylated at the delta-NH2 group of Orn by a Gly-Gly dipeptide. In a significant proportion (about 23%) of total muropeptides, the N-terminal Gly is substituted by a residue of phenylacetic acid. This is the first time phenylacetic acid is described as a component of bacterial murein. Possible implications for murein physiology and biosynthesis are discussed. Murein cross-linking is mediated by D-Ala-Gly-Gly peptide cross-bridges. Glycan chains are apparently terminated by (1-->6) anhydro N-acetylmuramic acid residues. Neither reducing sugars nor murein-bound macromolecules were detected. Murein from T. thermophilus presents an intermediate complexity between those of gram-positive and gram-negative organisms. The murein composition and peptide cross-bridges of T. thermophilus are typical for a gram-positive bacterium. However, the murein content, degree of cross-linkage, and glycan chain length for T. thermophilus are closer to those for gram-negative organisms and could explain the gram-negative character of Thermus spp.     

Phosphoenolpyruvate-insensitive phosphofructokinase isozyme from Thermus thermophilus HB8.

In the previous paper [Xu, J., Oshima, T., & Yoshida, M. (1990) J. Mol. Biol. 215, 597-606], we reported that phosphofructokinase from Thermus thermophilus is allosterically inhibited by phosphoenolpyruvate, which induces dissociation of the active four-subunit enzyme into an inactive two-subunit form. When T. thermophilus was cultured in a glucose-containing medium, another phosphofructokinase (PFK2) appeared in addition to the reported one (PFK1). The molecular weights of the native PFK2 molecule (132,000) and its subunit (34,500), which are slightly smaller than those of PFK1, suggest that PFK2 is also composed of four identical subunits. However, the hyperbolic kinetics and molecular form of PFK2 are not affected at all by phosphoenolpyruvate. The NH2-terminal amino acid sequences of subunits of PFK1 and PFK2 revealed that they are composed of very similar but different polypeptides.     

Crystal structure of the conserved protein TT1542 from Thermus thermophilus HB8

The TT1542 protein from Thermus thermophilus HB8 is annotated as a conserved hypothetical protein, and belongs to the DUF158 family in the Pfam database. A BLAST search revealed that homologs of TT1542 are present in a wide range of organisms. The TT1542 homologs in eukaryotes, PIG-L in mammals, and GPI12 in yeast and protozoa, have N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI) de-N-acetylase activity. Although most of the homologs in prokaryotes are hypothetical and have no known function, Rv1082 and Rv1170 from Mycobacterium tuberculosis are enzymes involved in the mycothiol detoxification pathway. Here we report the crystal structure of the TT1542 protein at 2.0 A resolution, which represents the first structure for this superfamily of proteins. The structure of the TT1542 monomer consists of a twisted beta-sheet composed of six parallel beta-strands and one antiparallel beta-strand (with the strand order 3-2-1-4-5-7-6) sandwiched between six alpha-helices. The N-terminal five beta-strands and four alpha-helices form an incomplete Rossmann fold-like structure. The structure shares some similarity to the sugar-processing enzymes with Rossmann fold-like domains, especially those of the GPGTF (glycogen phosphorylase/glycosyl transferase) superfamily, and also to the NAD(P)-binding Rossmann fold domains. TT1542 is a homohexamer in the crystal and in solution, the six monomers forming a cylindrical structure. Putative active sites are suggested by the structure and conserved amino acid residues.