Crystal structure of GTP-dependent dephospho-coenzyme A kinase from the hyperthermophilic archaeon, Thermococcus kodakarensis.

The biosynthesis pathways of coenzyme A (CoA) in most archaea involve several unique enzymes including dephospho-CoA kinase (DPCK) that converts dephospho-CoA to CoA in the final step of CoA biosynthesis in all domains of life. The archaeal DPCK is unrelated to the analogous bacterial and eukaryotic enzymes and shows no significant sequence similarity to any proteins with known structures. Unusually, the archaeal DPCK utilizes GTP as the phosphate donor although the analogous bacterial and eukaryotic enzymes are ATP-dependent kinases.

Crystal structure of the GH-46 subclass III chitosanase from Bacillus circulans MH-K1 in complex with chitotetraose.

Background: Chitosanases (EC 3.2.1.

Description of peptide bond planarity from high-resolution neutron crystallography.

Neutron crystallography is a highly effective method for visualizing hydrogen atoms in proteins. In our recent study, we successfully determined the high-resolution (1.2 Ã…) neutron structure of high-potential iron-sulfur protein, refining the coordinates of some amide protons without any geometric restraints.

Structural basis of the 24B3 antibody against the toxic conformer of amyloid β with a turn at positions 22 and 23.

Amyloid β-protein (Aβ) oligomers are involved in the early stages of Alzheimer's disease (AD) and antibodies against these toxic oligomers could be useful for accurate diagnosis of AD. We identified the toxic conformer of Aβ42 with a turn at positions 22/23, which has a propensity to form toxic oligomers. The antibody 24B3, developed by immunization of a toxic conformer surrogate E22P-Aβ9-35 in mice, was found to be useful for AD diagnosis using human cerebrospinal fluid (CSF).

Structures of oxygen dissociation intermediates of 400 kDa V2 hemoglobin provide coarse snapshots of the protein allostery.

Ever since the historic discovery of the cooperative oxygenation of its multiple subunits, hemoglobin (Hb) has been among the most exhaustively studied allosteric proteins. However, the lack of structural information on the intermediates between oxygenated and deoxygenated forms prevents our detailed understanding of the molecular mechanism of its allostery. It has been difficult to prepare crystals of intact oxy-deoxy intermediates and to individually identify the oxygen saturation for each subunit.

Revisiting the concept of peptide bond planarity in an iron-sulfur protein by neutron structure analysis.

The planarity of the peptide bond is important for the stability and structure formation of proteins. However, substantial distortion of peptide bonds has been reported in several high-resolution structures and computational analyses. To investigate the peptide bond planarity, including hydrogen atoms, we report a 1.

Coarse snapshots of oxygen-dissociation intermediates of a giant hemoglobin elucidated by determining the oxygen saturation in individual subunits in the crystalline state.

Cooperative oxygen binding of hemoglobin (Hb) has been studied for over half a century as a representative example of the allostericity of proteins. The most important problem remaining to be solved is the lack of structural information on the intermediates between the oxygenated and deoxygenated forms. In order to characterize the intermediate structures, it is necessary to obtain intermediate-state crystals, determine their oxygen saturations and then determine the oxygen saturations of each of their constituent subunits, all of which are challenging issues even now.

Identification and Enzymatic Analysis of an Archaeal ATP-Dependent Serine Kinase from the Hyperthermophilic Archaeon .

Serine kinase catalyzes the phosphorylation of free serine (Ser) to produce -phosphoserine (Sep). An ADP-dependent Ser kinase in the hyperthermophilic archaeon Thermococcus kodakarensis (-SerK) is involved in cysteine (Cys) biosynthesis and most likely Ser assimilation. An ATP-dependent Ser kinase in the mesophilic bacterium Staphylococcus aureus is involved in siderophore biosynthesis.

Altering the Phosphorylation Position of Pyrophosphate-Dependent -Inositol-1-Kinase Based on Its Crystal Structure.

Most kinases utilize ATP as a phosphate donor and phosphorylate a wide range of phosphate acceptors. An alternative phosphate donor is inorganic pyrophosphate (PPi), which costs only 1/1000 of ATP. To develop a method to engineer PPi-dependent kinases, we herein aimed to alter the product of PPi-dependent -inositol kinase from d-inositol 1-phosphate to d-inositol 3-phosphate.

Split conformation of Chaetomium thermophilum Hsp104 disaggregase.

Hsp104 and its bacterial homolog ClpB form hexameric ring structures and mediate protein disaggregation. The disaggregated polypeptide is thought to thread through the central channel of the ring. However, the dynamic behavior of Hsp104 during disaggregation remains unclear.

Characterization of Class IB Terpene Synthase: The First Crystal Structure Bound with a Substrate Surrogate.

Terpene synthases (TS) are classified into two broad types, Class I and II, based on the chemical strategy for initial carbocation formation and motif sequences of the catalytic site. We have recently identified a new class of enzymes, Class IB, showing the acceptability of long (C-C) prenyl-diphosphates as substrates and no amino acid sequence homology with known TS. Conversion of long prenyl-diphosphates such as heptaprenyl-diphosphate (C) is unusual and has never been reported for Class I and II enzymes.

Structural Insight into [NiFe] Hydrogenase Maturation by Transient Complexes between Hyp Proteins.

[NiFe] hydrogenases catalyze reversible hydrogen production/consumption. The core unit of [NiFe] hydrogenase consists of a large and a small subunit. The active site of the large subunit of [NiFe] hydrogenases contains a NiFe(CN)CO cluster.

X-ray crystallographic studies on the hydrogen isotope effects of green fluorescent protein at sub-ångström resolutions.

Hydrogen atoms are critical to the nature and properties of proteins, and thus deuteration has the potential to influence protein function. In fact, it has been reported that some deuterated proteins show different physical and chemical properties to their protiated counterparts. Consequently, it is important to investigate protonation states around the active site when using deuterated proteins.

Crystal structure of pantoate kinase from Thermococcus kodakarensis.

The coenzyme A biosynthesis pathways in most archaea involve two unique enzymes, pantoate kinase and phosphopantothenate synthetase, to convert pantoate to 4'-phosphopantothenate. Here, we report the first crystal structure of pantoate kinase from the hyperthermophilic archaeon, Thermococcus kodakarensis and its complex with ATP and a magnesium ion. The electron density for the adenosine moiety of ATP was very weak, which most likely relates to its broad nucleotide specificity.

Characterization of perdeuterated high-potential iron-sulfur protein with high-resolution X-ray crystallography.

Perdeuteration in neutron crystallography is an effective method for determining the positions of hydrogen atoms in proteins. However, there is shortage of evidence that the high-resolution details of perdeuterated proteins are consistent with those of the nondeuterated proteins. In this study, we determined the X-ray structure of perdeuterated high-potential iron-sulfur protein (HiPIP) at a high resolution of 0.

Subatomic resolution X-ray structures of green fluorescent protein.

Green fluorescent protein (GFP) is a light-emitting protein that does not require a prosthetic group for its fluorescent activity. As such, GFP has become indispensable as a molecular tool in molecular biology. Nonetheless, there has been no subatomic elucidation of the GFP structure owing to the structural polymorphism around the chromophore.

Crystal structure of heme A synthase from .

Heme A is an essential cofactor for respiratory terminal oxidases and vital for respiration in aerobic organisms. The final step of heme A biosynthesis is formylation of the C-8 methyl group of heme molecule by heme A synthase (HAS). HAS is a heme-containing integral membrane protein, and its structure and reaction mechanisms have remained unknown.

X-ray structure analysis of bacteriorhodopsin at 1.3 Å resolution.

Bacteriorhodopsin (bR) of Halobacterium salinarum is a membrane protein that acts as a light-driven proton pump. bR and its homologues have recently been utilized in optogenetics and other applications. Although the structures of those have been reported so far, the resolutions are not sufficient for elucidation of the intrinsic structural features critical to the color tuning and ion pumping properties.

Co-translational folding of α-helical proteins: structural studies of intermediate-length variants of the λ repressor.

Unlabelled: Nascent polypeptide chains fold cotranslationally, but the atomic-level details of this process remain unknown. Here, we report crystallographic, modeling, and spectroscopic studies of intermediate-length variants of the λ repressor N-terminal domain. Although the ranges of helical regions of the half-length variant were almost identical to those of the full-length protein, the relative orientations of these helices in the intermediate-length variants differed.

Crystal structures of a [NiFe] hydrogenase large subunit HyhL in an immature state in complex with a Ni chaperone HypA.

Ni-Fe clusters are inserted into the large subunit of [NiFe] hydrogenases by maturation proteins such as the Ni chaperone HypA via an unknown mechanism. We determined crystal structures of an immature large subunit HyhL complexed with HypA from Structure analysis revealed that the N-terminal region of HyhL extends outwards and interacts with the Ni-binding domain of HypA. Intriguingly, the C-terminal extension of immature HyhL, which is cleaved in the mature form, adopts a β-strand adjacent to its N-terminal β-strands.

Crystal structures of an archaeal chitinase ChiD and its ligand complexes.

Chitinase D (designated as Pc-ChiD) was found in a hyperthermophilic archaeon, Pyrococcus chitonophagus (previously described as Thermococcus chitonophagus), that was isolated from media containing only chitin as carbon source. Pc-ChiD displays chitinase activity and is thermostable at temperatures up to 95°C, suggesting its potential for industrial use. Pc-ChiD has a secretion signal peptide and two chitin-binding domains (ChBDs) in the N-terminal domain.

Crystal structure and functional analysis of large-terpene synthases belonging to a newly found subclass.

Thousands of terpenes have been identified to date. However, only two classes of enzymes are known to be involved in their biosynthesis, and each class has characteristic amino-acid motifs. We recently identified a novel large-terpene (C/C/C) synthase, which shares no motifs with known enzymes.

Identification of a pyrophosphate-dependent kinase and its donor selectivity determinants.

Almost all kinases utilize ATP as their phosphate donor, while a few kinases utilize pyrophosphate (PPi) instead. PPi-dependent kinases are often homologous to their ATP-dependent counterparts, but determinants of their different donor specificities remain unclear. We identify a PPi-dependent member of the ribokinase family, which differs from known PPi-dependent kinases, and elucidate its PPi-binding mode based on the crystal structures.

Structure of a [NiFe] hydrogenase maturation protease HycI provides insights into its substrate selectivity.

The immature large subunit of [NiFe] hydrogenases undergoes C-terminal cleavage by a specific protease in the final step of the post-translational process before assembly with other subunits. It has been reported that the [NiFe] hydrogenase maturation protease HycI from Thermococcus kodakarensis (TkHycI) has the catalytic ability to target the membrane-bound hydrogenase large subunit MbhL from T. kodakarensis.