Serial femtosecond X-ray crystallography of an anaerobically formed catalytic intermediate of copper amine oxidase.

The mechanisms by which enzymes promote catalytic reactions efficiently through their structural changes remain to be fully elucidated. Recent progress in serial femtosecond X-ray crystallography (SFX) using X-ray free-electron lasers (XFELs) has made it possible to address these issues. In particular, mix-and-inject serial crystallography (MISC) is promising for the direct observation of structural changes associated with ongoing enzymic reactions.

Re-evaluation of protein neutron crystallography with and without X-ray/neutron joint refinement.

Protein neutron crystallography is a powerful technique to determine the positions of H atoms, providing crucial biochemical information such as the protonation states of catalytic groups and the geometry of hydrogen bonds. Recently, the crystal structure of a bacterial copper amine oxidase was determined by joint refinement using X-ray and neutron diffraction data sets at resolutions of 1.14 and 1.

Microcrystal preparation for serial femtosecond X-ray crystallography of bacterial copper amine oxidase.

Recent advances in serial femtosecond X-ray crystallography (SFX) using X-ray free-electron lasers have paved the way for determining radiation-damage-free protein structures under nonfreezing conditions. However, the large-scale preparation of high-quality microcrystals of uniform size is a prerequisite for SFX, and this has been a barrier to its widespread application. Here, a convenient method for preparing high-quality microcrystals of a bacterial quinoprotein enzyme, copper amine oxidase from Arthrobacter globiformis, is reported.

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.

Neutron crystallography of copper amine oxidase reveals keto/enolate interconversion of the quinone cofactor and unusual proton sharing.

Recent advances in neutron crystallographic studies have provided structural bases for quantum behaviors of protons observed in enzymatic reactions. Thus, we resolved the neutron crystal structure of a bacterial copper (Cu) amine oxidase (CAO), which contains a prosthetic Cu ion and a protein-derived redox cofactor, topa quinone (TPQ). We solved hitherto unknown structures of the active site, including a keto/enolate equilibrium of the cofactor with a nonplanar quinone ring, unusual proton sharing between the cofactor and the catalytic base, and metal-induced deprotonation of a histidine residue that coordinates to the Cu.

thermodynamic analysis of conformational change of the topaquinone cofactor in bacterial copper amine oxidase.

In the catalytic reaction of copper amine oxidase, the protein-derived redox cofactor topaquinone (TPQ) is reduced by an amine substrate to an aminoresorcinol form (TPQ), which is in equilibrium with a semiquinone radical (TPQ). The transition from TPQ to TPQ is an endothermic process, accompanied by a significant conformational change of the cofactor. We employed the humid air and glue-coating (HAG) method to capture the equilibrium mixture of TPQ and TPQ in noncryocooled crystals of the enzyme from and found that the equilibrium shifts more toward TPQ in crystals than in solution.

Characterization of auxiliary iron-sulfur clusters in a radical -adenosylmethionine enzyme PqqE from AM1.

PqqE is a radical -adenosyl-l-methionine (SAM) enzyme that catalyzes the initial reaction of pyrroloquinoline quinone (PQQ) biosynthesis. PqqE belongs to the SPASM (subtilosin/PQQ/anaerobic sulfatase/mycofactocin maturating enzymes) subfamily of the radical SAM superfamily and contains multiple FeS clusters. To characterize the FeS clusters in PqqE from AM1, Cys residues conserved in the N-terminal signature motif (CX CX C) and the C-terminal seven-cysteine motif (CX GX CX CX CX CX CX C; = an unspecified number) were individually or simultaneously mutated into Ser.

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.

PqqE from Methylobacterium extorquens AM1: a radical S-adenosyl-l-methionine enzyme with an unusual tolerance to oxygen.

Methylobacterium extorquens AM1 is an aerobic facultative methylotroph known to secrete pyrroloquinoline quinone (PQQ), a cofactor of a number of bacterial dehydrogenases, into the culture medium. To elucidate the molecular mechanism of PQQ biosynthesis, we are focusing on PqqE which is believed to be the enzyme catalysing the first reaction of the pathway. PqqE belongs to the radical S-adenosyl-l-methionine (SAM) superfamily, in which most, if not all, enzymes are very sensitive to dissolved oxygen and rapidly inactivated under aerobic conditions.

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.

High-throughput de novo screening of receptor agonists with an automated single-cell analysis and isolation system.

Reconstitution of signaling pathways involving single mammalian transmembrane receptors has not been accomplished in yeast cells. In this study, intact EGF receptor (EGFR) and a cell wall-anchored form of EGF were co-expressed on the yeast cell surface, which led to autophosphorylation of the EGFR in an EGF-dependent autocrine manner. After changing from EGF to a conformationally constrained peptide library, cells were fluorescently labeled with an anti-phospho-EGFR antibody.

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.

High-resolution crystal structure of copper amine oxidase from Arthrobacter globiformis: assignment of bound diatomic molecules as O2.

The crystal structure of a copper amine oxidase from Arthrobacter globiformis was determined at 1.08 Å resolution with the use of low-molecular-weight polyethylene glycol (LMW PEG; average molecular weight ∼200) as a cryoprotectant. The final crystallographic R factor and Rfree were 13.

An automated system for high-throughput single cell-based breeding.

When establishing the most appropriate cells from the huge numbers of a cell library for practical use of cells in regenerative medicine and production of various biopharmaceuticals, cell heterogeneity often found in an isogenic cell population limits the refinement of clonal cell culture. Here, we demonstrated high-throughput screening of the most suitable cells in a cell library by an automated undisruptive single-cell analysis and isolation system, followed by expansion of isolated single cells. This system enabled establishment of the most suitable cells, such as embryonic stem cells with the highest expression of the pluripotency marker Rex1 and hybridomas with the highest antibody secretion, which could not be achieved by conventional high-throughput cell screening systems (e.

Electroporation and use of hepatitis B virus envelope L proteins as bionanocapsules.

Hepatitis B virus (HBV) envelope L proteins, when synthesized in yeast cells, form a hollow bionanocapsule (BNC) in which genes (including large plasmids up to 40 kbp), small interfering RNA (siRNA), drugs, and proteins can be enclosed by electroporation. BNCs made from L proteins have several advantages as a delivery system: Because they display a human liver-specific receptor (the pre-S region of the L protein) on their surface, BNCs can efficiently and specifically deliver their contents to human liver-derived cells and tissues ex vivo (in cell culture) and in vivo (in a mouse xenograft model). Retargeting can be achieved simply by substituting other biorecognition molecules such as antibodies, ligands, receptors, and homing peptides for the pre-S region.

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.

Hepatitis B virus envelope L protein-derived bio-nanocapsules: mechanisms of cellular attachment and entry into human hepatic cells.

A bio-nanocapsule (BNC) is a hollow nanoparticle consisting of an approximately 100-nm-diameter liposome with about 110 molecules of hepatitis B virus (HBV) surface antigen L protein embedded as a transmembrane protein. BNC can encapsulate various drugs and genes and deliver them specifically to human hepatic cells based on the ability of HBV to recognize human hepatocyte, which is integrated in the N-terminal region of L protein. However, it is elusive whether the cellular attachment and entry into hepatic cells of BNC utilize the early infection mechanism of HBV.

Structural insights into the substrate specificity of bacterial copper amine oxidase obtained by using irreversible inhibitors.

Copper amine oxidases (CAOs) catalyse the oxidation of various aliphatic amines to the corresponding aldehydes, ammonia and hydrogen peroxide. Although CAOs from various organisms share a highly conserved active-site structure including a protein-derived cofactor, topa quinone (TPQ), their substrate specificities differ considerably. To obtain structural insights into the substrate specificity of a CAO from Arthrobacter globiformis (AGAO), we have determined the X-ray crystal structures of AGAO complexed with irreversible inhibitors that form covalent adducts with TPQ.

Fluorophore-labeled nanocapsules displaying IgG Fc-binding domains for the simultaneous detection of multiple antigens.

Simultaneous detection of multiple antigens by conventional immunological methods has been limited by the source of primary antibodies. Each antibody should be derived from a different host species (or subclass of immunoglobulin (Ig)) for suppressing the cross-reactions of secondary antibodies. Here we describe an innovative method for simultaneous, rapid, and sensitive detection of multiple antigens using ∼30-nm bio-nanocapsules (BNCs) displaying IgG Fc-binding Z domains derived from Staphylococcus aureus protein A (ZZ-BNC).

Efficient and rapid purification of drug- and gene-carrying bio-nanocapsules, hepatitis B virus surface antigen L particles, from Saccharomyces cerevisiae.

Bio-nanocapsules (BNCs) are hollow particles (approx. 50 nm diameter) consisting of hepatitis B virus surface antigen (HBsAg) large (L, pre-S1+pre-S2+S) proteins embedded in a unilamellar liposome, sharing the same transmembrane S region with an immunogen of hepatitis B vaccine (i.e.

Production of H2S by 3-mercaptopyruvate sulphurtransferase.

Hydrogen sulfide (H(2)S) has been established as the third gaseous signaling molecule following nitric oxide and carbon monoxide and participates in a variety of cellular functions such as modulation of neuronal transmission, endothelium-dependent vasorelaxation, stimulation of angiogenesis and regulation of insulin release. Although cystathionine β-synthase and cystathionine γ-lyase have been regarded as the main producers of H(2)S in many tissues including brain, liver and kidney, Kimura and his colleagues have recently communicated that 3-mercaptopyruvate sulphurtransferase coupled with cysteine (aspartate) aminotransferase is responsible for the production of H(2)S in the vascular endothelium of the thoracic aorta [Shibuya et al. (2009) J.