High-resolution structure and biochemical properties of the LH1-RC photocomplex from the model purple sulfur bacterium, Allochromatium vinosum.

The mesophilic purple sulfur phototrophic bacterium Allochromatium (Alc.) vinosum (bacterial family Chromatiaceae) has been a favored model for studies of bacterial photosynthesis and sulfur metabolism, and its core light-harvesting (LH1) complex has been a focus of numerous studies of photosynthetic light reactions. However, despite intense efforts, no high-resolution structure and thorough biochemical analysis of the Alc. vinosum LH1 complex have been reported. Here we present cryo-EM structures of the Alc. vinosum LH1 complex associated with reaction center (RC) at 2.24 Å resolution. The overall structure of the Alc. vinosum LH1 resembles that of its moderately thermophilic relative Alc. tepidum in that it contains multiple pigment-binding α- and ß-polypeptides. Unexpectedly, however, six Ca ions were identified in the Alc. vinosum LH1 bound to certain α1/ß1- or α1/ß3-polypeptides through a different Ca2+-binding motif from that seen in Alc. tepidum and other Chromatiaceae that contain Ca2+-bound LH1 complexes. Two water molecules were identified as additional Ca2+-coordinating ligands. Based on these results, we reexamined biochemical and spectroscopic properties of the Alc. vinosum LH1-RC. While modest but distinct effects of Ca2+ were detected in the absorption spectrum of the Alc. vinosum LH1 complex, a marked decrease in thermostability of its LH1-RC complex was observed upon removal of Ca2+. The presence of Ca2+ in the photocomplex of Alc. vinosum suggests that Ca2+-binding to LH1 complexes may be a common adaptation in species of Chromatiaceae for conferring spectral and thermal flexibility on this key component of their photosynthetic machinery.

Modification of Silver Nanoplates with Cell-Binding Subunit of Bacterial Toxin and Their Antimicrobial Activity against Intracellular Bacteria.

Intracellular bacteria are able to survive and grow in host cells and often cause serious infectious diseases. The B subunit of the subtilase cytotoxin (SubB) found in enterohemorrhagic Escherichia coli O113:H21 recognizes sialoglycans on cell surfaces and triggers the uptake of cytotoxin by the cells, meaning that Sub B is a ligand molecule that is expected to be useful for drug delivery into cells. In this study, we conjugated SubB to silver nanoplates (AgNPLs) for use as an antibacterial drug and examined their antimicrobial activity against intracellularly infecting Salmonella typhimurium (S. typhimurium). The modification of AgNPLs with SubB improved their dispersion stability and antibacterial activity against planktonic S. typhimurium. The SubB modification enhanced the cellular uptake of AgNPLs, and intracellularly infecting S. typhimurium were killed at low concentrations of AgNPLs. Interestingly, larger amounts of SubB-modified AgNPLs were taken up by infected cells compared with uninfected cells. These results suggest that the S. typhimurium infection activated the uptake of the nanoparticles into the cells. SubB-modified AgNPLs are expected to be useful bactericidal systems for intracellularly infecting bacteria.

Structural Determination of the Nanocomplex of Borate with Styrene-Maleic Acid Copolymer-Conjugated Glucosamine Used as a Multifunctional Anticancer Drug.

The development of effective anticancer drugs is essential for chemotherapy that specifically targets cancer tissues. We recently synthesized a multifunctional water-soluble anticancer polymer drug consisting of styrene-maleic acid copolymer (SMA) conjugated with glucosamine and boric acid (BA) (SGB complex). It demonstrated about 10 times higher tumor-selective accumulation compared with accumulation in normal tissues because of the enhanced permeability and retention effect, and it inhibited tumor growth via glycolysis inhibition, mitochondrial damage, and thermal neutron irradiation. Gaining insight into the anticancer effects of this SGB complex requires a determination of its structure. We therefore investigated the chemical structure of the SGB complex by means of nuclear magnetic resonance, infrared (IR) spectroscopy, and liquid chromatography-mass spectrometry. To establish the chemical structure of the SGB complex, we synthesized a simple model compound─maleic acid-glucosamine (MAG) conjugate─by using a maleic anhydride (MA) monomer unit instead of the SMA polymer. We obtained two MAG-BA complexes (MAGB) with molecular weights of 325 and 343 after the MAG reaction with BA. We confirmed, by using IR spectroscopy, that MAGB formed a stable complex via an amide bond between MA and glucosamine and that BA bound to glucosamine via a diol bond. As a result of this chemical design, identified via analysis of MAGB, the SGB complex can release BA and demonstrate toxicity to cancer cells through inhibition of lactate secretion in mild hypoxia that mimics the tumor microenvironment. For clinical application of the SGB complex, we confirmed that this complex is stable in the presence of serum. These findings confirm that our design of the SGB complex has various advantages in targeting solid cancers and exerting therapeutic effects when combined with neutron irradiation.

Ectopic PTH-producing parathyroid cyst inside the thymus: a case report.

BACKGROUND: The hallmark of hyperparathyroidism is hypersecretion of parathyroid hormone (PTH) which results in hypercalcemia and hypophosphatemia. While hypercalcemia due to malignancy is often brought about by PTH-related protein in adults, PTH-producing tumors are quite rare in clinical practice. Additionally, from the point of embryology, it is very difficult to examine ectopic PTH-producing tissue such as ectopic parathyroid glands. Furthermore, clear histopathological criteria are not present. CASE PRESENTATION: A 57-year-old woman was referred to our hospital for hypercalcemia. Her parathyroid hormone (PTH) level was elevated, but there were no enlarged parathyroid glands. Although 99mTc-MIBI confirmed a localized and slightly hyperfunctioning parathyroid tissue in the anterior mediastinum, it was not typical as hyperfunctioning parathyroid. We finally diagnosed her as ectopic PTH-producing cyst-like tumor with venous sampling of PTH. She underwent anterosuperior mediastinal ectopic PTH-producing cyst-like tumor resection. It is noted that intact-PTH concentration of the fluid in the cyst was very high (19,960,000 pg/mL). Based on histopathological findings, we finally diagnosed her as ectopic PTH-producing parathyroid cyst inside the thymus. After resection of anterosuperior mediastinal thymus including ectopic PTH-producing parathyroid cyst, calcium and intact-PTH levels were decreased, and this patient was discharged without any sequelae. CONCLUSIONS: We should know the possibility of superior mediastinal ectopic PTH-producing parathyroid cyst inside the thymus among subjects with ectopic PTH-producing parathyroid glands. Particularly when the cyst is present in the superior mediastinum, it is necessary to do careful diagnosis based on not only positive but also negative findings in 99mTc-MIBI. It is noted that the patient's bloody fluid in the cyst contained 19,960,000 pg/mL of intact-PTH, and its overflow into blood stream resulted in hyperparathyroidism and hypercalcemia. Moreover, in such cases, the diagnosis is usually confirmed after through histological examination of ectopic PTH-producing parathyroid glands. We think that it is very meaningful to let clinicians know this case.

Controlled Delivery of an Anti-Inflammatory Toxin to Macrophages by Mutagenesis and Nanoparticle Modification.

Advances in drug delivery systems (DDSs) have enabled the specific delivery of drugs to target cells. Subtilase cytotoxin (SubAB) produced by certain enterohemorrhagic Escherichia coli strains induces endoplasmic reticulum (ER) stress and suppresses nitric oxide generation in macrophages. We previously reported that modification of SubAB with poly(D,L-lactide-co-glycolic) acid (PLGA) nanoparticles (SubAB-PLGA NPs) increased intracellular uptake of SubAB and had an anti-inflammatory effect on macrophages. However, specific delivery of SubAB to macrophages could not be achieved because its effects on other cell types were not negligible. Therefore, to suppress non-specific SubAB binding, we used low-binding mutant SubABS35A (S35A) in which the 35th serine of the B subunit was mutated to alanine. In a macrophage cell line, PLGA NPs modified with S35A (S35A-PLGA NPs) induced ER stress and had anti-inflammatory effects similar to WT-PLGA NPs. However, in an epithelial cell line, S35A-PLGA NPs induced lower ER stress than WT-PLGA NPs. These results suggest that S35A is selectively delivered to macrophages rather than epithelial cells by modification with PLGA NPs and exerts anti-inflammatory effects. Our findings provide a useful technique for protein delivery to macrophages and encourage medical applications of DDSs for the treatment of inflammatory diseases.

The GTP responsiveness of PI5P4Kß evolved from a compromised trade-off between activity and specificity.

Unlike most kinases, phosphatidylinositol 5-phosphate 4-kinase ß (PI5P4Kß) utilizes GTP as a physiological phosphate donor and regulates cell growth under stress (i.e., GTP-dependent stress resilience). However, the genesis and evolution of its GTP responsiveness remain unknown. Here, we reveal that PI5P4Kß has acquired GTP preference by generating a short dual-nucleotide-recognizing motif called the guanine efficient association (GEA) motif. Comparison of nucleobase recognition with 660 kinases and 128 G proteins has uncovered that most kinases and PI5P4Kß use their main-chain atoms for adenine recognition, while the side-chain atoms are required for guanine recognition. Mutational analysis of the GEA motif revealed that the acquisition of GTP reactivity is accompanied by an extended activity toward inosine triphosphate (ITP) and xanthosine triphosphate (XTP). Along with the evolutionary analysis data that point to strong negative selection of the GEA motif, these results suggest that the GTP responsiveness of PI5P4Kß has evolved from a compromised trade-off between activity and specificity, underpinning the development of the GTP-dependent stress resilience.

Weakly Acidic pH and Reduction Dual Stimuli-Responsive Gel Particles.

This paper reports the facile preparation of dual stimuli-responsive gel particles that simultaneously respond to weakly acidic and reducing stimuli and the application of these gel particles as a drug delivery carrier. The dual stimuli-responsive gel particles composed of a pH-responsive polymer network cross-linked with reduction stimuli-responsive disulfide cross-links, and biocompatible poly(ethylene glycol) cross-links were prepared by soap-free emulsion polymerization. The resulting gel particles were colloidally stable at physiological ionic strength and had a diameter of approximately 200 nm with a narrow size distribution. The resulting gel particles slightly swelled in an acidic environment. On the other hand, the gel particles drastically swelled under simultaneous weakly acidic and reducing conditions because of the ionization of tertiary amino groups in the gel network and a decrease in the cross-linking density resulting from cleavage of the disulfide cross-links. When cells were treated with the gel particles, they were taken up by cells via the endocytosis pathway and distributed in the cytosol after endosomal escape by the proton sponge effect. In addition, a hydrophobic drug, doxorubicin (Dox), was loaded into the gel particles through hydrophobic interactions. Dox was released from the gel particles under weakly acidic and reducing conditions, while the Dox release was inhibited at neutral pH. The weakly acidic pH- and reduction stimuli-responsive release of Dox from gel particles was attributed to the drastic swelling of these particles. The fascinating properties of the dual stimuli-responsive gel particles suggest that they can provide a useful platform for designing intracellular drug delivery carriers.

EPR-Effect Enhancers Strongly Potentiate Tumor-Targeted Delivery of Nanomedicines to Advanced Cancers: Further Extension to Enhancement of the Therapeutic Effect.

For more than three decades, enhanced permeability and retention (EPR)-effect-based nanomedicines have received considerable attention for tumor-selective treatment of solid tumors. However, treatment of advanced cancers remains a huge challenge in clinical situations because of occluded or embolized tumor blood vessels, which lead to so-called heterogeneity of the EPR effect. We previously developed a method to restore impaired blood flow in blood vessels by using nitric oxide donors and other agents called EPR-effect enhancers. Here, we show that two novel EPR-effect enhancers-isosorbide dinitrate (ISDN, Nitrol®) and sildenafil citrate-strongly potentiated delivery of three macromolecular drugs to tumors: a complex of poly(styrene-co-maleic acid) (SMA) and cisplatin, named Smaplatin® (chemotherapy); poly(N-(2-hydroxypropyl)methacrylamide) polymer-conjugated zinc protoporphyrin (photodynamic therapy and imaging); and SMA glucosamine-conjugated boric acid complex (boron neutron capture therapy). We tested these nanodrugs in mice with advanced C26 tumors. When these nanomedicines were administered together with ISDN or sildenafil, tumor delivery and thus positive therapeutic results increased two- to four-fold in tumors with diameters of 15 mm or more. These results confirmed the rationale for using EPR-effect enhancers to restore tumor blood flow. In conclusion, all EPR-effect enhancers tested showed great potential for application in cancer therapy.

Structural and Functional Analyses of the Tridomain-Nonribosomal Peptide Synthetase FmoA3 for 4-Methyloxazoline Ring Formation.

Nonribosomal peptide synthetases (NRPSs) are attractive targets for bioengineering to generate useful peptides. FmoA3 is a single modular NRPS composed of heterocyclization (Cy), adenylation (A), and peptidyl carrier protein (PCP) domains. It uses α-methyl-l-serine to synthesize a 4-methyloxazoline ring, probably with another Cy domain in the preceding module FmoA2. Here, we determined the head-to-tail homodimeric structures of FmoA3 by X-ray crystallography (apo-form, with adenylyl-imidodiphosphate and α-methyl-l-seryl-AMP) and cryogenic electron microscopy single particle analysis, and performed site-directed mutagenesis experiments. The data revealed that α-methyl-l-serine can be accommodated in the active site because of the extra space around Ala688. The Cy domains of FmoA2 and FmoA3 catalyze peptide bond formation and heterocyclization, respectively. FmoA3's Cy domain seems to lose its donor PCP binding activity. The collective data support a proposed catalytic cycle of FmoA3.

Polymer-conjugated glucosamine complexed with boric acid shows tumor-selective accumulation and simultaneous inhibition of glycolysis.

We synthesized unique water-soluble synthetic-polymer, styrene-maleic acid copolymer (SMA) conjugated glucosamine (SG); which formed a stable complex with boric acid (BA). This complex had a mean particle size of 15 nm by light scattering, and single peak in gel permeation chromatography. The particles were taken up by tumor cells five times faster than free BA in vitro and liberated BA at acidic tumor pH (5-7). Liberated BA inhibited glycolysis and resulted in tumor suppression in vivo. Intravenously injected SGB-complex did bind with albumin, and plasma half-life was about 8 h in mice, and accumulated to tumor tissues about 10 times more than in normal organs. IC50 of SGB-complex for HeLa cells under pO2 of 6-9% was about 20 µg/ml (free BA equivalent), 150 times more potent than free BA. Neutron irradiation of human oral cancer cells with SGB-complex resulted in 16 times greater cell-killing than that without SGB-complex. In vivo antitumor effect was evaluated after neutron irradiation only once in SCC VII tumor bearing mice and significant tumor suppression was confirmed. These results indicate that SGB-complex is a unique multifunctional anticancer agent with much more potent activity under low pO2 conditions as in large advanced cancers.

GZD824 Inhibits GCN2 and Sensitizes Cancer Cells to Amino Acid Starvation Stress.

Eukaryotic initiation factor 2α (eIF2α) kinase general control nonderepressible 2 (GCN2) drives cellular adaptation to amino acid limitation by activating the integrated stress response that induces activating transcription factor 4 (ATF4). Here, we found that a multikinase inhibitor, GZD824, which we identified using a cell-based assay with ATF4 immunostaining, inhibited the GCN2 pathway in cancer cells. Indeed, GZD824 suppressed GCN2 activation, eIF2α phosphorylation, and ATF4 induction during amino acid starvation stress. However, at lower nonsuppressive concentrations, GZD824 paradoxically stimulated eIF2α phosphorylation and ATF4 expression in a GCN2-dependent manner under unstressed conditions. Such dual properties conceivably arose from a direct effect on GCN2, as also observed in a cell-free GCN2 kinase assay and shared by a selective GCN2 inhibitor. Consistent with the GCN2 pathway inhibition, GZD824 sensitized certain cancer cells to amino acid starvation stress similarly to ATF4 knockdown. These results establish GZD824 as a multikinase GCN2 inhibitor and may enhance its utility as a drug under development. SIGNIFICANCE STATEMENT: GZD824, as a direct general control nonderepressible 2 (GCN2) inhibitor, suppresses activation of the integrated stress response during amino acid limitation, whereas it paradoxically stimulates this stress-signaling pathway at lower nonsuppressive concentrations. The pharmacological activity we identify herein will provide the basis for the use of GZD824 to elucidate the regulatory mechanisms of GCN2 and to evaluate the potential of the GCN2-activating transcription factor 4 pathway as a target for cancer therapy.

Preparation of Biodegradable PLGA-Nanoparticles Used for pH-Sensitive Intracellular Delivery of an Anti-inflammatory Bacterial Toxin to Macrophages.

Poly(D,L-lactide-co-glycolic) acid (PLGA) is a synthetic copolymer that has been used to design micro/nanoparticles as a carrier for macromolecules, such as protein and nucleic acids, that can be internalized by the endocytosis pathway. However, it is difficult to control the intracellular delivery to target organelles. Here we report an intracellular delivery system of nanoparticles modified with bacterial cytotoxins to the endoplasmic reticulum (ER) and anti-inflammatory activity of the nanoparticles. Subtilase cytotoxin (SubAB) is a bacterial toxin in certain enterohemorrhagic Escherichia coli (EHEC) strains that cleaves the host ER chaperone BiP and suppresses nuclear factor-kappaB (NF-κB) activation and nitric oxide (NO) generation in macrophages at sub-lethal concentration. PLGA-nanoparticles were modified with oligo histidine-tagged (6 × His-tagged) recombinant SubAB (SubAB-PLGA) through a pH-sensitive linkage, and their translocation to the ER in macrophage cell line J774.1 cells, effects on inducible NO synthase (iNOS), and levels of tumor necrosis factor (TNF)-α cytokine induced by lipopolysaccharide (LPS) were examined. Compared with free SubAB, SubAB-PLGA was significantly effective in BiP cleavage and the induction of the ER stress marker C/EBP homologous protein (CHOP) in J774.1 cells. Furthermore, SubAB-PLGA attenuated LPS-stimulated induction of iNOS and TNF-α. Our findings provide useful information for protein delivery to macrophages and may encourage therapeutic applications of nanoparticles to the treatment of inflammatory diseases.

Involvement of protein disulfide isomerase in subtilase cytotoxin-induced cell death in HeLa cells.

Subtilase cytotoxin (SubAB) is a member of bacterial AB5 toxin produced by certain enterohemorrhagic E. coli strains which cleaves host chaperone BiP in endoplasmic reticulum (ER), leading to ER stress-mediated cytotoxicity. Previous study suggested that protein disulfide isomerase (PDI), an enzyme which catalyzes the formation and breakage of disulfide bonds in proteins, regulates AB5 toxin such as cholera toxin by unfolding of A subunit, leading to its translocation into cytosol to induce disease. Although SubAB targets ER and has similar A subunit to that of other AB5 toxins, it is unclear whether PDI can modulate the SubAB function. Here we determined the role of PDI on SubAB-induced BiP cleavage, ER stress response and cytotoxicity in HeLa cells. We found that PDI knockdown significantly suppressed SubAB-induced BiP cleavage and eIF2α phosphorylation. The accumulation of SubAB in ER was perturbed upon PDI knockdown. Finally, cell viability assay showed that PDI knockdown and PDI inhibitor canceled the SubAB-induced cytotoxicity. Present results suggested that SubAB, after cellular uptake, translocates into ER and interacts with BiP that might be modulated by PDI. Identification of pivotal role of host proteins on bacterial toxin to elicit its pathogenesis is necessary basis for development of potential chemotherapy and new diagnostic strategy for control of toxin-producing bacterial infections.

Gold Coating of Silver Nanoplates for Enhanced Dispersion Stability and Efficient Antimicrobial Activity against Intracellular Bacteria.

Silver nanoparticles have antibacterial activity. However, the nanoparticles are unstable and easily form aggregates, which decreases their antibacterial activity. To improve the dispersion stability of silver nanoparticles in aqueous media and to increase their effectiveness as antibacterial agents, we coated triangular plate-like silver nanoparticles (silver nanoplates, Ag NPLs) with one or two layers of gold atoms (Ag@Au1L NPLs and Ag@Au2L NPLs, respectively). These gold coatings improved the dispersion stability in aqueous media with high salt concentrations. Ag@Au1L NPLs showed stronger antibacterial activity on pathogenic bacteria than Ag NPLs and Ag@Au2L NPLs. Furthermore, the Ag@Au1L NPLs decreased the number of bacteria in RAW 264.7 cells. The Ag@Au1L NPLs displayed no cytotoxicity towards RAW 264.7 cells and could be used as antibacterial agents for intracellular bacterial infections.

Characterization of Heme Orientational Disorder in a Myoglobin Reconstituted with a Trifluoromethyl-Group-Substituted Heme Cofactor.

The orientation of a CF3-substituted heme in sperm whale myoglobin and L29F, H64L, L29F/H64Q, and H64Q variant proteins has been investigated using 19F NMR spectroscopy to elucidate structural factors responsible for the thermodynamic stability of the heme orientational disorder, i.e., the presence of two heme orientations differing by a 180° rotation about the 5-15 meso axis, with respect to the protein moiety. Crystal structure of the met-aquo form of the wild-type myoglobin reconstituted with 13,17-bis(2-carboxylatoethyl)-3,8-diethyl-2,12,18-trimethyl-7-trifluoromethylporphyrinatoiron(III), determined at resolution of 1.25 Å, revealed the presence of the heme orientational disorder. Alterations of the salt bridge between the heme 13-propionate and Arg45(CD3) side chains due to the mutations resulted in equilibrium constants of the heme orientational disorder ranging between 0.42 and 1.4. Thus, the heme orientational disorder is affected by the salt bridge associated with the heme 13-propionate side chain, confirming the importance of the salt bridge in the heme binding to the protein.

The crystal structure of a new O-demethylase from Sphingobium sp. strain SYK-6.

In the cell, tetrahydrofolate (H4 folate) derivatives with a C1 unit are utilized in various ways, such as for the synthesis of amino acids and nucleic acids. While H4 folate derivatives with the C1 unit are typically produced in the glycine cleavage system, Sphingobium sp. strain SYK-6, which can utilize lignin-derived aromatic compounds as a sole source of carbon and energy, lacks this pathway, probably due to its unique nutrient requirements. In this bacterium, H4 folate-dependent O-demethylases in catabolic pathways for lignin-derived aromatic compounds seem to be involved in the C1 metabolism. LigM is one of the O-demethylases and catalyzes a C1-unit transfer from vanillate (VNL) to H4 folate. As the primary structure of LigM shows a similarity to T-protein in the glycine cleavage system, we hypothesized that LigM has evolved from T-protein, acquiring its unique biochemical and biological functions. To prove this hypothesis, structure-based understanding of its catalytic reaction is essential. Here, we determined the crystal structure of LigM in apo form and in complex with substrates and H4 folate. These crystal structures showed that the overall structure of LigM is similar to T-protein, but LigM has a few distinct characteristics, particularly in the active site. Structure-based mutational analysis revealed that His60 and Tyr247, which are not conserved in T-protein, are essential to the catalytic activity of LigM and their interactions with the oxygen atom in the methoxy group of VNL seem to facilitate a methyl moiety (C1-unit) transfer to H4 folate. Taken together, our structural data suggest that LigM has evolved divergently from T-protein. DATABASES: All atomic coordinates of the crystal structures determined in this study have been deposited to PDB. LigM: 5X1I, LigM-VNL complex: 5X1J, LigM-3-O-methylgallate complex: 5X1K, LigM-H4 folate complex: 5X1IL, LigM-H4 folate-protocatechuate (PCA) complex (P21 21 2): 5X1M, LigM-H4 folate-PCA complex (P31 21): 5X1N.

Overcoming a hemihedral twinning problem in tetrahydrofolate-dependent O-demethylase crystals by the microseeding method.

A tetrahydrofolate-dependent O-demethylase, LigM, from Sphingobium sp. SYK-6 was crystallized by the hanging-drop vapour-diffusion method. However, the obtained P3121 or P3221 crystals, which diffracted to 2.5-3.3 Šresolution, were hemihedrally twinned. To overcome the twinning problem, microseeding using P3121/P3221 crystals as microseeds was performed with optimization of the reservoir conditions. As a result, another crystal form was obtained. The newly obtained crystal diffracted to 2.5-3.0 Šresolution and belonged to space group P21212, with unit-cell parameters a = 102.0, b = 117.3, c = 128.1 Å. The P21212 crystals diffracted to better than 2.0 Šresolution after optimizing the cryoconditions. Phasing using the single anomalous diffraction method was successful at 3.0 Šresolution with a Pt-derivative crystal. This experience suggested that microseeding is an effective method to overcome the twinning problem, even when twinned crystals are utilized as microseeds.

Complete pyridine-nucleotide-specific conversion of an NADH-dependent ferredoxin reductase.

The coenzyme specificity of enzymes is one of the critical parameters for the engineered production of biological compounds using bacteria. Since NADPH is produced abundantly in photosynthetic organisms, conversion of an NADH-specific enzyme into an NADPH-specific one is a useful approach for the efficient carbon-neutral production of biological compounds in photosynthetic organisms. In the present study, an NADH-specific ferredoxin reductase component, BphA4 of biphenyl dioxygenase BphA from Acidovorax sp. strain KKS102, was changed to an NADPH-dependent form using a method combining structure-based systematic mutations and site-directed random mutagenesis. The resultant CRG mutant, in which Glu175-Thr176-Gln177 of an NADH-recognition loop in the wild-type BphA4 was replaced with Cys175-Arg176-Gly177, was highly specific and active for NADPH, and its biochemical and structural properties for NADPH were nearly the same as those of the wild-type BphA4 for NADH. In addition, this mutation project was assessed by a semi-empirical prediction method of mutation effects, and the results suggested that the CRG mutant was one of the best NADPH-specific mutants.

Structural basis of the γ-lactone-ring formation in ascorbic acid biosynthesis by the senescence marker protein-30/gluconolactonase.

The senescence marker protein-30 (SMP30), which is also called regucalcin, exhibits gluconolactonase (GNL) activity. Biochemical and biological analyses revealed that SMP30/GNL catalyzes formation of the γ-lactone-ring of L-gulonate in the ascorbic acid biosynthesis pathway. The molecular basis of the γ-lactone formation, however, remains elusive due to the lack of structural information on SMP30/GNL in complex with its substrate. Here, we report the crystal structures of mouse SMP30/GNL and its complex with xylitol, a substrate analogue, and those with 1,5-anhydro-D-glucitol and D-glucose, product analogues. Comparison of the crystal structure of mouse SMP30/GNL with other related enzymes has revealed unique characteristics of mouse SMP30/GNL. First, the substrate-binding pocket of mouse SMP30/GNL is designed to specifically recognize monosaccharide molecules. The divalent metal ion in the active site and polar residues lining the substrate-binding cavity interact with hydroxyl groups of substrate/product analogues. Second, in mouse SMP30/GNL, a lid loop covering the substrate-binding cavity seems to hamper the binding of L-gulonate in an extended (or all-trans) conformation; L-gulonate seems to bind to the active site in a folded conformation. In contrast, the substrate-binding cavities of the other related enzymes are open to the solvent and do not have a cover. This structural feature of mouse SMP30/GNL seems to facilitate the γ-lactone-ring formation.

In vivo gene transfection via intravitreal injection of cationic liposome/plasmid DNA complexes in rabbits.

To optimize the in vivo ocular transfection efficiency of plasmid DNA (pDNA)/cationic liposome complexes, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)/dioleoylphosphatidylethanolamine (DOPE) (1:1 molar ratio) liposomes and DOTMA/cholesterol (Chol) (1:1 molar ratio) liposomes were prepared with varying amounts of pDNA. pDNA/cationic liposome complexes were intravitreally injected (100 microL) in rabbits, and luciferase activity in the cornea, aqueous humor, iris-ciliary body, lens, vitreous body, and retina was measured. Transfection efficiency of pDNA alone did not change with pDNA ranging from 40 to 85 mg. In contrast, transfection efficiency of pDNA complexed with DOTMA/Chol liposomes significantly increased with the amount of pDNA ranging from 40 to 85 microg (P < 0.05). pDNA complexed with DOTMA/DOPE liposomes could not be prepared with pDNA greater than 60 microg. Among these experiments, pDNA (85 microg) complexed with DOTMA/Chol liposomes (pDNA:cationic liposome charge ratio (- : +) = 1.0:2.0) showed the highest transfection efficiency in the ocular tissue and its transfection-mediated luciferase activity peaked at 3 days. Among the ocular tissues, the highest gene expression was observed in the aqueous humor.