Shigenori Nishimura

Structural analysis of lipocalin-type prostaglandin D synthase complexed with biliverdin by small-angle X-ray scattering and multi-dimensional NMR.

Lipocalin-type prostaglandin D synthase (L-PGDS) acts as both a PGD(2) synthase and an extracellular transporter for small lipophilic molecules. From a series of biochemical studies, it has been found that L-PGDS has an ability to bind a variety of lipophilic ligands such as biliverdin, bilirubin and retinoids in vitro. Therefore, we considered that it is necessary to clarify the molecular structure of L-PGDS upon binding ligand in order to understand the physiological relevance of L-PGDS as a transporter protein. We investigated a molecular structure of L-PGDS/biliverdin complex by small-angle X-ray scattering (SAXS) and multi-dimensional NMR measurements, and characterized the binding mechanism in detail. SAXS measurements revealed that L-PGDS has a globular shape and becomes compact by 1.3A in radius of gyration on binding biliverdin. NMR experiments revealed that L-PGDS possessed an eight-stranded antiparallel beta-barrel forming a central cavity. Upon the titration with biliverdin, some cross-peaks for residues surrounding the cavity and EF-loop and H2-helix above the beta-barrel shifted, and the intensity of other cross-peaks decreased with signal broadenings in (1)H-(15)N heteronuclear single quantum coherence spectra. These results demonstrate that L-PGDS holds biliverdin within the beta-barrel, and the conformation of the loop regions above the beta-barrel changes upon binding biliverdin. Through such a conformational change, the whole molecule of L-PGDS becomes compact.

Kinetic and crystallographic analyses of the catalytic domain of chitinase from Pyrococcus furiosus- the role of conserved residues in the active site.

The hyperthermostable chitinase from the hyperthermophilic archaeon Pyrococcusu2003furiosus has a unique multidomain structure containing two chitin‐binding domains and two catalytic domains, and exhibits strong crystalline chitin hydrolyzing activity at high temperature. In order to investigate the structure–function relationship of this chitinase, we analyzed one of the catalytic domains (AD2) using mutational and kinetic approaches, and determined the crystal structure of AD2 complexed with chito‐oligosaccharide substrate. Kinetic studies showed that, among the acidic residues in the signature sequence of familyu200318 chitinases (DXDXE motif), the second Asp (D2) and Glu (E) residues play critical roles in the catalysis of archaeal chitinase. Crystallographic analyses showed that the side‐chain of the catalytic proton‐donating E residue is restrained into the favorable conformer for proton donation by a hydrogen bond interaction with the adjacent D2 residue. The comparison of active site conformations of familyu200318 chitinases provides a new criterion for the subclassification of familyu200318 chitinase based on the conformational change of the D2 residue.

Drug delivery system for poorly water-soluble compounds using lipocalin-type prostaglandin D synthase.

Lipocalin-type prostaglandin D synthase (L-PGDS) is a member of the lipocalin superfamily and a secretory lipid-transporter protein, which binds a wide variety of hydrophobic small molecules. Here we show the feasibility of a novel drug delivery system (DDS), utilizing L-PGDS, for poorly water-soluble compounds such as diazepam (DZP), a major benzodiazepine anxiolytic drug, and 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione (NBQX), an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist and anticonvulsant. Calorimetric experiments revealed for both compounds that each L-PGDS held three molecules with high binding affinities. By mass spectrometry, the 1:3 complex of L-PGDS and NBQX was observed. L-PGDS of 500μM increased the solubility of DZP and NBQX 7- and 2-fold, respectively, compared to PBS alone. To validate the potential of L-PGDS as a drug delivery vehicle in vivo, we have proved the prospective effects of these compounds via two separate delivery strategies. First, the oral administration of a DZP/L-PGDS complex in mice revealed an increased duration of pentobarbital-induced loss of righting reflex. Second, the intravenous treatment of ischemic gerbils with NBQX/L-PGDS complex showed a protective effect on delayed neuronal cell death at the hippocampal CA1 region. We propose that our novel DDS could facilitate pharmaceutical development and clinical usage of various water-insoluble compounds.

Thermal unfolding mechanism of lipocalin-type prostaglandin D synthase

Lipocalin‐type prostaglandin (PG)u2003D synthase (L‐PGDS) is a dual‐functioning protein in the lipocalin family, acting as a PGD2‐synthesizing enzyme and as an extracellular transporter for small lipophilic molecules. We earlier reported that denaturant‐induced unfolding of L‐PGDS follows a four‐state pathway, including an activity‐enhanced state and an inactive intermediate state. In this study, we investigated the thermal unfolding mechanism of L‐PGDS by using differential scanning calorimetry (DSC) and CD spectroscopy. DSC measurements revealed that the thermal unfolding of L‐PGDS was a completely reversible process at pHu20034.0. The DSC curves showed no concentration dependency, demonstrating that the thermal unfolding of L‐PGDS involved neither intermolecular interaction nor aggregation. On the basis of a simple two‐state unfolding mechanism, the ratio of van’t Hoff enthalpy (ΔHvH) to calorimetric enthalpy (ΔHcal) was below 1, indicating the presence of an intermediate state (I) between the native state (N) and unfolded state (U). Then, statistical thermodynamic analyses of a three‐state unfolding process were performed. The heat capacity curves fit well with a three‐state process; and the estimated transition temperature (Tm) and enthalpy change (ΔHcal) of the N↔I and I↔U transitions were 48.2u2003°C and 190u2003kJ·mol−1, and 60.3u2003°C and 144u2003kJ·mol−1, respectively. Correspondingly, the thermal unfolding monitored by CD spectroscopy at 200, 235 and 290u2003nm revealed that L‐PGDS unfolded through the intermediate state, where its main chain retained the characteristic β‐sheet structure without side‐chain interactions.

Kinetically trapped metastable intermediate of a disulfide-deficient mutant of the starch-binding domain of glucoamylase

Refolding of a thermally unfolded disulfide‐deficient mutant of the starch‐binding domain of glucoamylase was investigated using differential scanning calorimetry, isothermal titration calorimetry, CD, and 1H NMR. When the protein solution was rapidly cooled from a higher temperature, a kinetic intermediate was formed during refolding. The intermediate was unexpectedly stable compared with typical folding intermediates that have short half‐lives. It was shown that this intermediate contained substantial secondary structure and tertiary packing and had the same binding ability with β‐cyclodextrin as the native state, suggesting that the intermediate is highly‐ordered and native‐like on the whole. These characteristics differ from those of partially folded intermediates such as molten globule states. Far‐UV CD spectra showed that the secondary structure was once disrupted during the transition from the intermediate to the native state. These results suggest that the intermediate could be an off‐pathway type, possibly a misfolded state, that has to undergo unfolding on its way to the native state.

Backbone chemical shifts assignments, secondary structure, and ligand binding of a family GH-19 chitinase from moss, Bryum coronatum

Family GH19 chitinases have been recognized as important in the plant defense against fungal pathogens. However, their substrate-recognition mechanism is still unknown. We report here the first resonance assignment of NMR spectrum of a GH19 chitinase from moss, Bryum coronatum (BcChi-A). The backbone signals were nearly completely assigned, and the secondary structure was estimated based on the chemical shift values. The addition of the chitin dimer to the enzyme solution perturbed the chemical shifts of HSQC resonances of the amino acid residues forming the putative substrate-binding cleft. Further NMR analysis of the ligand binding to BcChi-A will improve understanding of the substrate-recognition mechanism of GH-19 enzymes.

Mechanism of chitosan recognition by CBM32 carbohydrate-binding modules from a Paenibacillus sp. IK-5 chitosanase/glucanase

An antifungal chitosanase/glucanase isolated from the soil bacterium Paenibacillus sp. IK-5 has two CBM32 chitosan-binding modules (DD1 and DD2) linked in tandem at the C-terminus. In order to obtain insights into the mechanism of chitosan recognition, the structures of DD1 and DD2 were solved by NMR spectroscopy and crystallography. DD1 and DD2 both adopted a β-sandwich fold with several loops in solution as well as in crystals. On the basis of chemical shift perturbations in(1)H-(15)N-HSQC resonances, the chitosan tetramer (GlcN)4 was found to bind to the loop region extruded from the core β-sandwich of DD1 and DD2. The binding site defined by NMR in solution was consistent with the crystal structure of DD2 in complex with (GlcN)3, in which the bound (GlcN)3 stood upright on its non-reducing end at the binding site. Glu(14)of DD2 appeared to make an electrostatic interaction with the amino group of the non-reducing end GlcN, and Arg(31), Tyr(36)and Glu(61)formed several hydrogen bonds predominantly with the non-reducing end GlcN. No interaction was detected with the reducing end GlcN. Since Tyr(36)of DD2 is replaced by glutamic acid in DD1, the mutation of Tyr(36)to glutamic acid was conducted in DD2 (DD2-Y36E), and the reverse mutation was conducted in DD1 (DD1-E36Y). Ligand-binding experiments using the mutant proteins revealed that this substitution of the 36th amino acid differentiates the binding properties of DD1 and DD2, probably enhancing total affinity of the chitosanase/glucanase toward the fungal cell wall.

Characterization of the novel Trypanosoma brucei inosine 5 '-monophosphate dehydrogenase

There is an alarming rate of human African trypanosomiasis recrudescence in many parts of sub-Saharan Africa. Yet, the disease has no successful chemotherapy. Trypanosoma lacks the enzymatic machinery for the de novo synthesis of purine nucleotides, and is critically dependent on salvage mechanisms. Inosine 5-monophosphate dehydrogenase (IMPDH) is responsible for the rate-limiting step in guanine nucleotide metabolism. Here, we characterize recombinant Trypanosoma brucei IMPDH (TbIMPDH) to investigate the enzymatic differences between TbIMPDH and host IMPDH. Size-exclusion chromatography and analytical ultracentrifugation sedimentation velocity experiments reveal that TbIMPDH forms a heptamer, different from type 1 and 2 mammalian tetrameric IMPDHs. Kinetic analysis reveals calculated K m values of 30 and 1300 μ m for IMP and NAD, respectively. The obtained K m value of TbIMPDH for NAD is approximately 20-200-fold higher than that of mammalian enzymes and indicative of a different NAD binding mode between trypanosomal and mammalian IMPDHs. Inhibition studies show K i values of 3·2 μ m, 21 nM and 3·3 nM for ribavirin 5-monophosphate, mycophenolic acid and mizoribine 5-monophosphate, respectively. Our results show that TbIMPDH is different from its mammalian counterpart and thus may be a good target for further studies on anti-trypanosomal drugs.

Interaction of a goose-type lysozyme with chitin oligosaccharides as determined by NMR spectroscopy.

The interaction between a goose-type lysozyme from ostrich egg white (OEL) and chitin oligosaccharides [(GlcNAc)(n) (n = 2, 4 and 6)] was studied by nuclear magnetic resonance (NMR) spectroscopy. A stable isotope-labelled OEL was produced in Pichia pastoris, and backbone resonance assignments for the wild-type and an inactive mutant (E73A OEL) were achieved using modern multi-dimensional NMR techniques. NMR titration was performed with (GlcNAc)(n) for mapping the interaction sites of the individual oligosaccharides based on the shifts in the two-dimensional heteronuclear single quantum correlation (HSQC) resonances. In wild-type OEL, the interaction sites for (GlcNAc)(n) were basically similar to those determined by X-ray crystallography. In E73A OEL, however, the interaction sites were spread more widely over the substrate-binding cleft than expected, due to the multiple modes of binding. The association constant for E73A OEL and (GlcNAc)(6) calculated from the shifts in the Asp97 resonance (7.2 × 10(3) M(-1)) was comparable with that obtained by isothermal titration calorimetry (5.3 × 10(3) M(-1)). The interaction was enthalpy-driven as judged from the thermodynamic parameters (ΔH = -6.1 kcal/mol and TΔS = -1.0 kcal/mol). This study provided novel insights into the oligosaccharide binding mechanism and the catalytic residues of the enzymes belonging to family GH-23.

Maltal Binding Mechanism and a Role of the Mobile Loop of Soybean β-Amylase

The inhibition of hydration of maltal (α-D-glucopyranosyl-(l→4)-2-deoxy-D-glucal) catalyzed by soybean ²-amylase with 4-0-α-D-glucopyranosyl-(l→4)-l-deoxynojirimycin (GDN) was investigated at 25°C and at pH 5.4. As the concentrations of GDN used were comparable to that of the enzyme, Hendersons treatment was applied to this system. It was found that two maltal molecules bind to the enzyme according to a random mechanism and GDN inhibits the hydration of maltal competitively at subsites 1 and 2, and noncompetitively at the other site. On the basis of this result, it was inferred that the role of the mobile loop of this enzyme is to create a convenient catalytic environment for the hydration, and the closing of the active site by the mobile loop is induced by the binding of maltal.