Eight genes are necessary and sufficient for biogenesis of quinohemoprotein amine dehydrogenase.

Quinohemoprotein amine dehydrogenase (QHNDH) containing a peptidyl quinone cofactor, cysteine tryptophylquinone, is produced in the periplasm of Gram-negative bacteria through an intricate process of post-translational modification that requires at least 8 genes including those encoding 3 nonidentical subunits and 3 modifying enzymes. Our heterologous expression study has revealed that the 8 genes are necessary and sufficient for the QHNDH biogenesis.

Functional and structural characterization of a flavoprotein monooxygenase essential for biogenesis of tryptophylquinone cofactor.

Bioconversion of peptidyl amino acids into enzyme cofactors is an important post-translational modification. Here, we report a flavoprotein, essential for biosynthesis of a protein-derived quinone cofactor, cysteine tryptophylquinone, contained in a widely distributed bacterial enzyme, quinohemoprotein amine dehydrogenase. The purified flavoprotein catalyzes the single-turnover dihydroxylation of the tryptophylquinone-precursor, tryptophan, in the protein substrate containing triple intra-peptidyl crosslinks that are pre-formed by a radical S-adenosylmethionine enzyme within the ternary complex of these proteins.

Cellular uptake of hepatitis B virus envelope L particles is independent of sodium taurocholate cotransporting polypeptide, but dependent on heparan sulfate proteoglycan.

Sodium taurocholate cotransporting polypeptide (NTCP) was recently discovered as a hepatitis B virus (HBV) receptor, however, the detailed mechanism of HBV entry is not yet fully understood. We investigated the cellular entry pathway of HBV using recombinant HBV surface antigen L protein particles (bio-nanocapsules, BNCs). After the modification of L protein in BNCs with myristoyl group, myristoylated BNCs (Myr-BNCs) were found to bind to NTCP in vitro, and inhibit in vitro HBV infection competitively, suggesting that Myr-BNCs share NTCP-dependent infection machinery with HBV.

Mutational analysis of hepatitis B virus pre-S1 (9-24) fusogenic peptide.

A hollow nanoparticle known as a bio-nanocapsule (BNC) consisting of hepatitis B virus (HBV) envelope L protein and liposome (LP) can encapsulate drugs and genes and thereby deliver them in vitro and in vivo to human hepatic tissues, specifically by utilizing the HBV-derived infection machinery. Recently, we identified a low pH-dependent fusogenic domain at the N-terminal part of the pre-S1 region of the HBV L protein (amino acid residues 9 to 24; NPLGFFPDHQLDPAFG), which shows membrane destabilizing activity (i.e.

Bio-nanocapsules displaying various immunoglobulins as an active targeting-based drug delivery system.

Unlabelled: The bio-nanocapsule (BNC) is an approximately 30-nm particle comprising the hepatitis B virus (HBV) envelope L protein and a lipid bilayer. The L protein harbors the HBV-derived infection machinery; therefore, BNC can encapsulate payloads such as drugs, nucleic acids, and proteins and deliver them into human hepatocytes specifically in vitro and in vivo. To diversify the possible functions of BNC, we generated ZZ-BNC by replacing the domain indispensable for the human hepatotrophic property of BNC (N-terminal region of L protein) with the tandem form of the IgG Fc-binding Z domain of Staphylococcus aureus protein A.

Cytokine-dependent activation of JAK-STAT pathway in Saccharomyces cerevisiae.

Protein phosphorylation is an important post-translational modification for intracellular signaling molecules, mostly found in serine and threonine residues. Tyrosine phosphorylations are very few events (less than 0.1% to phosphorylated serine/threonine residues), but capable of governing cell fate decisions involved in proliferation, differentiation, apoptosis, and oncogenic transformation.

Probing the Catalytic Mechanism of Copper Amine Oxidase from Arthrobacter globiformis with Halide Ions.

The catalytic reaction of copper amine oxidase proceeds through a ping-pong mechanism comprising two half-reactions. In the initial half-reaction, the substrate amine reduces the Tyr-derived cofactor, topa quinone (TPQ), to an aminoresorcinol form (TPQamr) that is in equilibrium with a semiquinone radical (TPQsq) via an intramolecular electron transfer to the active-site copper. We have analyzed this reductive half-reaction in crystals of the copper amine oxidase from Arthrobacter globiformis.

The Radical S-Adenosyl-L-methionine Enzyme QhpD Catalyzes Sequential Formation of Intra-protein Sulfur-to-Methylene Carbon Thioether Bonds.

The bacterial enzyme designated QhpD belongs to the radical S-adenosyl-L-methionine (SAM) superfamily of enzymes and participates in the post-translational processing of quinohemoprotein amine dehydrogenase. QhpD is essential for the formation of intra-protein thioether bonds within the small subunit (maturated QhpC) of quinohemoprotein amine dehydrogenase. We overproduced QhpD from Paracoccus denitrificans as a stable complex with its substrate QhpC, carrying the 28-residue leader peptide that is essential for the complex formation.

Identification of genes essential for the biogenesis of quinohemoprotein amine dehydrogenase.

The structural genes encoding quinohemoprotein amine dehydrogenase (QHNDH) in Gram-negative bacteria constitute a polycistronic operon together with several nearby genes, which are collectively termed "qhp". We previously showed that the qhpD gene, which lies between qhpA and qhpC (encoding the α and γ subunits of QHNDH, respectively), and the qhpE gene, which follows qhpB (encoding the β subunit), both encode enzymes specifically involved in the posttranslational modification of the γ subunit and are hence essential for QHNDH biogenesis in Paracoccus denitrificans [Ono, K., et al.

An unusual subtilisin-like serine protease is essential for biogenesis of quinohemoprotein amine dehydrogenase.

Quinohemoprotein amine dehydrogenase (QHNDH), an αβγ heterotrimer present in the periplasm of several Gram-negative bacteria, catalyzes the oxidative deamination of various aliphatic amines such as n-butylamine for assimilation as carbon and energy sources. The γ subunit of mature QHNDH contains a protein-derived quinone cofactor, cysteine tryptophylquinone, and three intrapeptidyl thioether cross-links between Cys and Asp or Glu residues. In its cytoplasmic nascent form, the γ subunit has a 28-residue N-terminal leader peptide that is necessary for the production of active QHNDH but must be removed in the following maturation process.

A protein-protein interaction map of trypanosome ~20S editosomes.

Mitochondrial mRNA editing in trypanosomatid parasites involves several multiprotein assemblies, including three very similar complexes that contain the key enzymatic editing activities and sediment at ~20S on glycerol gradients. These ~20S editosomes have a common set of 12 proteins, including enzymes for uridylyl (U) removal and addition, 2 RNA ligases, 2 proteins with RNase III-like domains, and 6 proteins with predicted oligonucleotide binding (OB) folds. In addition, each of the 3 distinct ~20S editosomes contains a different RNase III-type endonuclease, 1 of 3 related proteins and, in one case, an additional exonuclease.

Structural bases for the specific interactions between the E2 and E3 components of the Thermus thermophilus 2-oxo acid dehydrogenase complexes.

Pyruvate dehydrogenase (PDH), branched-chain 2-oxo acid dehydrogenase (BCDH) and 2-oxoglutarate dehydrogenase (OGDH) are multienzyme complexes that play crucial roles in several common metabolic pathways. These enzymes belong to a family of 2-oxo acid dehydrogenase complexes that contain multiple copies of three different components (E1, E2 and E3). For the Thermus thermophilus enzymes, depending on its substrate specificity (pyruvate, branched-chain 2-oxo acid or 2-oxoglutarate), each complex has distinctive E1 (E1p, E1b or E1o) and E2 (E2p, E2b or E2o) components and one of the two possible E3 components (E3b and E3o).

Structure of P-protein of the glycine cleavage system: implications for nonketotic hyperglycinemia.

The crystal structure of the P-protein of the glycine cleavage system from Thermus thermophilus HB8 has been determined. This is the first reported crystal structure of a P-protein, and it reveals that P-proteins do not involve the alpha(2)-type active dimer universally observed in the evolutionarily related pyridoxal 5'-phosphate (PLP)-dependent enzymes. Instead, novel alphabeta-type dimers associate to form an alpha(2)beta(2) tetramer, where the alpha- and beta-subunits are structurally similar and appear to have arisen by gene duplication and subsequent divergence with a loss of one active site.

Ligand-induced conformational changes and a reaction intermediate in branched-chain 2-oxo acid dehydrogenase (E1) from Thermus thermophilus HB8, as revealed by X-ray crystallography.

The alpha(2)beta(2) tetrameric E1 component of the branched-chain 2-oxo acid (BCOA) dehydrogenase multienzyme complex is a thiamin diphosphate (ThDP)-dependent enzyme. E1 catalyzes the decarboxylation of a BCOA concomitant with the formation of the alpha-carbanion/enamine intermediate, 2-(1-hydroxyalkyl)-ThDP, followed by transfer of the 1-hydroxyalkyl group to the distal sulfur atom on the lipoamide of the E2 component. In order to elucidate the catalytic mechanism of E1, the alpha- and beta-subunits of E1 from Thermus thermophilus HB8 have been co-expressed in Escherichia coli, purified and crystallized as a stable complex, and the following crystal structures have been analyzed: the apoenzyme (E1(apo)), the holoenzyme (E1(holo)), E1(holo) in complex with the substrate analogue 4-methylpentanoate (MPA) as an ES complex model, and E1(holo) in complex with 4-methyl-2-oxopentanoate (MOPA) as the alpha-carbanion/enamine intermediate (E1(ceim)).

Structure of Thermus thermophilus HB8 H-protein of the glycine-cleavage system, resolved by a six-dimensional molecular-replacement method.

The glycine-cleavage system is a multi-enzyme complex consisting of four different components (the P-, H-, T- and L-proteins). Recombinant H-protein corresponding to that from Thermus thermophilus HB8 has been overexpressed, purified and crystallized. Synchrotron radiation from BL44B2 at SPring-8 was used to collect a native data set to 2.

Coexpression, purification, crystallization and preliminary X-ray characterization of glycine decarboxylase (P-protein) of the glycine-cleavage system from Thermus thermophilus HB8.

Thermus thermophilus (Tth) HB8 glycine decarboxylase (P-protein) is an alpha(2)beta(2) tetrameric enzyme with a total molecular mass of 200 kDa. The alpha- and beta-subunits of the Tth P-protein have been coexpressed in Escherichia coli and purified as a stable complex. Dynamic light-scattering measurements indicated the recombinant protein to be monodisperse and its size to be consistent with an alpha(2)beta(2) tetrameric composition.

Mechanism of preferential enrichment, an unusual enantiomeric resolution phenomenon caused by polymorphic transition during crystallization of mixed crystals composed of two enantiomers.

The mechanism of Preferential Enrichment, an unusual enantiomeric resolution phenomenon observed upon recrystallization of a series of racemic crystals which are classified as a racemic mixed crystal with fairly ordered arrangement of the two enantiomers, has been studied. On the basis of the existence of polymorphs and the occurrence of the resulting polymorphic transition during crystallization from solution, the mechanism has been accounted for in terms of (1) a preferential homochiral molecular association to form one-dimensional chain structures in the supersaturated solution of the racemate or nonracemic sample with a low ee value, (2) a kinetic formation of a metastable crystalline phase retaining the homochiral chain structures in a process of nucleation, (3) a polymorphic transition from the metastable phase to a stable one followed by enantioselective liberation of the excess R (or S) enantiomers from the transformed crystal into solution at the beginning of crystal growth to result in a slight enrichment (up to 10% ee) of the opposite S (or R) enantiomer in the deposited crystals, together with an enantiomeric enrichment of the R (or S) enantiomer in the mother liquor, and (4) a chiral discrimination by the once formed S (or R)-rich stable crystalline phase in a process of the subsequent crystal growth, leading to a considerable enantiomeric enrichment of the R (or S) enantiomer up to 100% ee in the mother liquor. The processes (3) and (4) are considered to be directly responsible for an enrichment of one enantiomer in the mother liquor.

Extremely Long C-C Bond in (-)-trans-1,2-Di-tert-butyl-1,2-diphenyl- and 1,1-Di-tert-butyl-2,2-diphenyl-3,8-dichlorocyclobuta[b]naphthalenes.

The sterically bulky t-Bu-substituted derivatives of 1,1,2,2-tetraphenyl-3,8-dichlorobuta[b]naphthalene (5), trans-1,2-tert-butyl-1,2-diphenyl- (8) and 1,1-di-tert-butyl-2,2-diphenyl-3,8-dichlorocyclobuta[b]naphthalene (12), were prepared. X-ray analysis of 8 and 12 at 150 K showed that C-C bonds of 8 and 12 are 1.686 and 1.