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 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.     

Expression of 3-hydroxyisobutyrate dehydrogenase in cultured neural cells

The branched-chain amino acids (BCAAs) – isoleucine, leucine, and valine – belong to the limited group of substances transported through the blood–brain barrier. One of the functions they are thought to have in brain is to serve as substrates for meeting parenchymal energy demands. Previous studies have shown the ubiquitous expression of a branched-chain alpha-keto acid dehydrogenase among neural cells. This enzyme catalyzes the initial and rate-limiting step in the irreversible degradative pathway for the carbon skeleton of valine and the other two branched-chain amino acids. Unlike the acyl-CoA derivates in the irreversible part of valine catabolism, 3-hydroxyisobutyrate could be expected to be released from cells by transport across the mitochondrial and plasma membranes. This could indeed be demonstrated for cultured astroglial cells. Therefore, to assess the ability of neural cells to make use of this valine-derived carbon skeleton as a metabolic substrate for the generation of energy, we investigated the expression in cultured neural cells of the enzyme processing this hydroxy acid, 3-hydroxyisobutyrate dehydrogenase (HIBDH). To achieve this, HIBDH was purified from bovine liver to serve as antigen for the production of an antiserum. Affinity-purified antibodies against HIBDH specifically recognized the enzyme in liver and brain homogenates. Immunocytochemistry demonstrated the ubiquitous expression of HIBDH among cultured glial (astroglial, oligodendroglial, microglial, and ependymal cells) and neuronal cells. Using an RT-PCR technique, these findings were corroborated by the detection of HIBDH mRNA in these cells. Furthermore, immunofluorescence double-labeling of astroglial cells with antisera against HIBDH and the mitochondrial marker pyruvate dehydrogenase localized HIBDH to mitochondria. The expression of HIBDH in neural cells demonstrates their potential to utilize valine imported into the brain for the generation of energy.     

Crystal structure of a family 4 uracil-DNA glycosylase from Thermus thermophilus HB8

Uracil-DNA glycosylase (UDG; EC 3.2.2.-) removes uracil from DNA to initiate DNA base excision repair. Since hydrolytic deamination of cytosine to uracil is one of the most frequent DNA-damaging events in all cells, UDG is an essential enzyme for maintaining the integrity of genomic information. For the first time, we report the crystal structure of a family 4 UDG from Thermus thermophilus HB8 (TthUDG) complexed with uracil, solved at 1.5 angstroms resolution. As opposed to UDG enzymes in its other families, TthUDG possesses a [4Fe-4S] cluster. This iron-sulfur cluster, which is distant from the active site, interacts with loop structures and has been suggested to be unessential to the activity but necessary for stabilizing the loop structures. In addition to the iron-sulfur cluster, salt-bridges and ion pairs on the molecular surface and the presence of proline on loops and turns is thought to contribute to the enzyme's thermostability. Despite very low levels of sequence identity with Escherichia coli and human UDGs (family 1) and E.coli G:T/U mismatch-specific DNA glycosylase (MUG) (family 2), the topology and order of secondary structures of TthUDG are similar to those of these distant relatives. Furthermore, the coordinates of the core structure formed by beta-strands are almost the same. Positive charge is distributed over the active-site groove, where TthUDG would bind DNA strands, as do UDG enzymes in other families. TthUDG recognizes uracil specifically in the same manner as does human UDG (family 1), rather than guanine in the complementary strand DNA, as does E.coli MUG (family 2). These results suggest that the mechanism by which family 4 UDGs remove uracils from DNA is similar to that of family 1 enzymes.     

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.     

Crystal structure of a novel zinc-binding ATP sulfurylase from Thermus thermophilus HB8

ATP sulfurylase (ATPS) is a ubiquitous enzyme that catalyzes the transfer of the adenylyl group from ATP to inorganic sulfate, producing adenosine 5'-phosphosulfate (APS) and pyrophosphate. The crystal structure of ATPS from Thermus thermophilus HB8 (TtATPS, 347 amino acid residues) in complex with APS was determined at 2.5 A resolution. TtATPS is composed of three domains [domain I (residues 1-134), domain II (residues 135-290), and domain III (residues 291-347)], like the Riftia pachyptila symbiont ATPS, but lacks a fourth domain present in ATPSs from the yeast Saccharomyces cerevisiae and from the fungus Penicillium chrysogenum. TtATPS forms a dimer in the crystal, and the manner of subunit association is different from that observed in dimeric R. pachyptila symbiont ATPS and in the hexameric S. cerevisiae and P. chrysogenum ATPSs. APS is located in the active site of TtATPS, which contains several motifs (QXRN, HXXH, and GRD) conserved in ATPSs. Unexpectedly, TtATPS binds one metal ion per subunit in domain III. XAFS measurement of the crystal and the Bijvoet difference Fourier map unambiguously characterized the metal ion as a zinc ion. The zinc ion is tetrahedrally coordinated by Cys294, Cys297, Cys306, and His310, and could not be removed from the protein by treatment with EDTA. The zinc ion binding site is far from the active site. Because all four residues coordinated to the zinc ion are conserved in the ATPSs from thermophilic bacteria such as Archaeoglobus fulgidus, Pyrococcus abyssi, and Sulfolobus solfataricus, zinc ion chelation may contribute to the thermal stability of these ATPSs.     

Crystal structure of a lysine biosynthesis enzyme,LysX, from Thermus thermophilus HB8

The thermophilic bacterium Thermus thermophilus synthesizes lysine through the alpha-aminoadipate pathway, which uses alpha-aminoadipate as a biosynthetic intermediate of lysine. LysX is the essential enzyme in this pathway, and is believed to catalyze the acylation of alpha-aminoadipate. We have determined the crystal structures of LysX and its complex with ADP at 2.0A and 2.38A resolutions, respectively. LysX is composed of three alpha+beta domains, each composed of a four to five-stranded beta-sheet core flanked by alpha-helices. The C-terminal and central domains form an ATP-grasp fold, which is responsible for ATP binding. LysX has two flexible loop regions, which are expected to play an important role in substrate binding and protection. In spite of the low level of sequence identity, the overall fold of LysX is surprisingly similar to that of other ATP-grasp fold proteins, such as D-Ala:D-Ala ligase, PurT-encoded glycinamide ribonucleotide transformylase, glutathione synthetase, and synapsin I. In particular, they share a similar spatial arrangement of the amino acid residues around the ATP-binding site. This observation strongly suggests that LysX is an ATP-utilizing enzyme that shares a common evolutionary ancestor with other ATP-grasp fold proteins possessing a carboxylate-amine/thiol ligase activity.     

Crystal structure of the 2'-5' RNA ligase from Thermus thermophilus HB8

The 2'-5' RNA ligase family members are bacterial and archaeal RNA ligases that ligate 5' and 3' half-tRNA molecules with 2',3'-cyclic phosphate and 5'-hydroxyl termini, respectively, to the product containing the 2'-5' phosphodiester linkage. Here, the crystal structure of the 2'-5' RNA ligase protein from an extreme thermophile, Thermus thermophilus HB8, was solved at 2.5A resolution. The structure of the 2'-5' RNA ligase superimposes well on that of the Arabidopsis thaliana cyclic phosphodiesterase (CPDase), which hydrolyzes ADP-ribose 1",2"-cyclic phosphate (a product of the tRNA splicing reaction) to the monoester ADP-ribose 1"-phosphate. Although the sequence identity between the two proteins is remarkably low (9.3%), the 2'-5' RNA ligase and CPDase structures have two HX(T/S)X motifs in their corresponding positions. The HX(T/S)X motifs play important roles in the CPDase activity, and are conserved in both the CPDases and 2'-5' RNA ligases. Therefore, the catalytic mechanism of the 2'-5' RNA ligase may be similar to that of the CPDase. On the other hand, the electrostatic potential of the cavity of the 2'-5' RNA ligase is positive, but that of the CPDase is negative. Furthermore, in the CPDase, two loops with low B-factors cover the cavity. In contrast, in the 2'-5' RNA ligase, the corresponding loops form an open conformation and are flexible. These characteristics may be due to the differences in the substrates, tRNA and ADP-ribose 1",2"-cyclic phosphate.     

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.     

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.     

ATP-induced structural change of dephosphocoenzyme A kinase from Thermus thermophilus HB8

Dephosphocoenzyme A kinase (DCK) catalyzes phosphorylation in the final step of coenzyme A (CoA) biosynthesis. In this phosphorylation process, domain movements play a very important role. To reveal the structural changes induced by ligand binding, we determined the crystal structure of DCK from Thermus thermophilus HB8 by the multiwavelength anomalous dispersion method at 2.8 A. The crystal structure includes three independent protein molecules in the asymmetric unit: One is a liganded form and the others are unliganded. The topology shows a canonical nucleotide-binding protein possessing the P-loop motif. A structure homology search by DALI revealed the similarity of the DCKs from T. thermophilus HB8, Haemophilus influenzae, and Escherichia coli. Structural comparisons between the liganded and unliganded forms of DCK from T. thermophilus HB8 indicated domain movements induced by adenosine triphosphate (ATP) binding. For the domain movements, proline residues confer flexibility at the domain linkages. In particular, Pro91 plays an important role in moving the CoA domain.     

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.     

Crystal structure of phenylacetic acid degradation protein PaaG from Thermus thermophilus HB8

Microbial degradation of phenylacetic acid proceeds via the hybrid pathway that includes formation of a coenzyme A thioester, ring hydroxylation, non‐oxygenolytic ring opening, and β‐oxidation‐like reactions. A phenylacetic acid degradation protein PaaG is a member of the crotonase superfamily, and is a candidate non‐oxygenolytic ring‐opening enzyme. The crystal structure of PaaG from Thermus thermophilus HB8 was determined at a resolution of 1.85 Å. PaaG consists of three identical subunits related by local three‐fold symmetry. The monomer is comprised of a spiral and a helical domain with a fold characteristic of the crotonase superfamily. A putative active site residue, Asp136, is situated in an active site cavity and surrounded by several hydrophobic and hydrophilic residues. The active site cavity is sufficiently large to accommodate a ring substrate. Two conformations are observed for helix H2 located adjacent to the active site. Helix H2 is kinked at Asn81 in two subunits, whereas it is kinked at Leu77 in the other subunit, and the side chain of Tyr80 is closer to Asp136. This indicates that catalytic reaction of PaaG may proceed with large conformational changes at the active site. Asp136 is the only conserved polar residue in the active site. It is located at the same position as those of 4‐chlorobenzoyl‐CoA dehalogenase and peroxisomal Δ³,Δ²‐enoyl‐CoA isomerase, indicating that PaaG may undergo isomerization or a ring‐opening reaction via a Δ³,Δ²‐enoyl‐CoA isomerase‐like mechanism. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Crystal structure of inorganic pyrophosphatase from Thermus thermophilus.

The 3-dimensional structure of inorganic pyrophosphatase from Thermus thermophilus (T-PPase) has been determined by X-ray diffraction at 2.0 A resolution and refined to R = 15.3%. The structure consists of an antiparallel closed beta-sheet and 2 alpha-helices and resembles that of the yeast enzyme in spite of the large difference in size (174 and 286 residues, respectively), little sequence similarity beyond the active center (about 20%), and different oligomeric organization (hexameric and dimeric, respectively). The similarity of the polypeptide folding in the 2 PPases provides a very strong argument in favor of an evolutionary relationship between the yeast and bacterial enzymes. The same Greek-key topology of the 5-stranded beta-barrel was found in the OB-fold proteins, the bacteriophage gene-5 DNA-binding protein, toxic-shock syndrome toxin-1, and the major cold-shock protein of Bacillus subtilis. Moreover, all known nucleotide-binding sites in these proteins are located on the same side of the beta-barrel as the active center in T-PPase. Analysis of the active center of T-PPase revealed 17 residues of potential functional importance, 16 of which are strictly conserved in all sequences of soluble PPases. Their possible role in the catalytic mechanism is discussed on the basis of the present crystal structure and with respect to site-directed mutagenesis studies on the Escherichia coli enzyme. The observed oligomeric organization of T-PPase allows us to suggest a possible mechanism for the allosteric regulation of hexameric PPases.