Characterization of the extracellular domain of sensor histidine kinase NagS from Paenibacillus sp. str. FPU-7: nagS interacts with oligosaccharide binding protein NagB1 in complexes with N, N'-diacetylchitobiose.

We have previously isolated the Gram-positive chitin-degrading bacterium Paenibacillus sp. str. FPU-7. This bacterium traps chitin disaccharide (GlcNAc)2 on its cell surface using two homologous solute-binding proteins, NagB1 and NagB2. Bacteria use histidine kinase (HK) of the two-component regulatory system as an extracellular environment sensor. In this study, we found that nagS, which encodes a HK, is located next to the nagB1 gene. Biochemical experiments revealed that the NagS sensor domain (NagS30-294) interacts with the NagB1-(GlcNAc)2 complex. However, proof of NagS30-294 interacting with NagB1 without (GlcNAc)2 is currently unavailable. In contrast to NagB1, no complex formation was observed between NagS30-294 and NagB2, even in the presence of (GlcNAc)2. The NagS30-294 crystal structure at 1.8 Å resolution suggested that the canonical tandem-Per-Arnt-Sim fold recognizes the NagB1-(GlcNAc)2 complex. This study provides insight into the recognition of chitin oligosaccharides by bacteria.

Mutagenesis and structure-based analysis of the role of Tryptophan525 of γ-glutamyltranspeptidase from Pseudomonas nitroreducens.

γ-Glutamyltranspeptidase (GGT) is a ubiquitous enzyme that catalyzes the hydrolysis of the γ-glutamyl linkage of γ-glutamyl compounds and the transfer of their γ-glutamyl moiety to acceptor substrates. Pseudomonas nitroreducens GGT (PnGGT) is used for the industrial synthesis of theanine, thus it is important to determine the structural basis of hydrolysis and transfer reactions and identify the acceptor site of PnGGT to improve the efficient of theanine synthesis. Our previous structural studies of PnGGT have revealed that crucial interactions between three amino acid residues, Trp385, Phe417, and Trp525, distinguish PnGGT from other GGTs. Here we report the role of Trp525 in PnGGT based on site-directed mutagenesis and structural analyses. Seven mutant variants of Trp525 were produced (W525F, W525V, W525A, W525G, W525S, W525D, and W525K), with substitution of Trp525 by nonaromatic residues resulting in dramatically reduced hydrolysis activity. All Trp525 mutants exhibited significantly increased transfer activity toward hydroxylamine with hardly any effect on acceptor substrate preference. The crystal structure of PnGGT in complex with the glutamine antagonist, 6-diazo-5-oxo-l-norleucine, revealed that Trp525 is a key residue limiting the movement of water molecules within the PnGGT active site.

Crystal structure of the catalytic unit of thermostable GH87 α-1,3-glucanase from Streptomyces thermodiastaticus strain HF3-3.

α-1,3-Glucan is a homopolymer composed of D-glucose (Glc) and it is an extracellular polysaccharide found in dental plaque due to Streptococcus species. α-1,3-Glucanase from Streptomyces thermodiastaticus strain HF3-3 (Agl-ST) has been identified as a thermostable α-1,3-glucanase, which is classified into glycoside hydrolase family 87 (GH87) and specifically hydrolyzes α-1,3-glucan with an endo-action. The enzyme has a potential to inhibit the production of dental plaque and to be used for biotechnological applications. Here we show the structure of the catalytic unit of Agl-ST determined at 1.16 Å resolution using X-ray crystallography. The catalytic unit is composed of two modules, a ß-sandwich fold module, and a right-handed ß-helix fold module, which resembles other structural characterized GH87 enzymes from Bacillus circulans str. KA-304 and Paenibacillus glycanilyticus str. FH11, with moderate sequence identities between each other (approximately 27% between the catalytic units). However, Agl-ST is smaller in size and more thermally stable than the others. A disulfide bond that anchors the C-terminal coil of the ß-helix fold, which is expected to contribute to thermal stability only exists in the catalytic unit of Agl-ST.

Immunochromatographic detection of human hemoglobin from deteriorated bloodstains due to methamphetamine contamination, aging, and heating.

Japanese police conduct highly sensitive and quick blood tests to detect human hemoglobin (Hb), because bloodstains left at a crime scene have probative value of circumstantial evidence in a criminal investigation. Although DNA detection from a bloodstain is a useful tool to identify an individual, doing so requires evidence that the bloodstain is of human origin. Stimulant drug abuse and dependence causes major social problems and crimes in Japan, and bloodstains are often found inside syringes seized from drug abusers. In this case, Hb often cannot be detected by conventional testing as high concentrations of stimulants, such as methamphetamine hydrochloride (MA), in blood trigger polymerization of Hb molecules, which become insoluble under non-reducing conditions and can no longer be detected by immunochromatographic detection kits. To overcome this problem, we analyzed methods to detect denatured Hb from bloodstains contaminated with MA. Reduction of polymerized Hb with a strong denaturing agent was required to solubilize polymers into monomers, suggesting that Hb aggregation is caused by aberrant formation of disulfide bonds. Based on these results, we established a pretreatment method, called Fukui's Reduction and Eiken's Dilution (FRED), that enables highly sensitive detection of human Hb from bloodstains mixed with MA by reducing and refolding of denatured Hb. This powerful method can be applied to blood that has been boiled or has otherwise deteriorated for over 20 years.

Crystal structure analysis and enzymatic characterization of γ-glutamyltranspeptidase from Pseudomonas nitroreducens.

Theanine (γ-glutamylethylamide) is an amino acid analog that reduces blood pressure and improves immune responses. The ϒ-glutamyltranspeptidase (GGT) from Pseudomonas nitroreducens IFO12694 (PnGGT) has a unique preference for primary amines as ϒ-glutamyl acceptors over standard L-amino acids and peptides. This characteristic is useful for the synthesis of theanine. We used X-ray crystallographic analysis to understand the structural basis of PnGGT's hydrolysis and transpeptidation reactions and to characterize its previously unidentified acceptor site. Structural studies of PnGGT have shown that key interactions between three residues (Trp385, Phe417, and Trp525) distinguish PnGGT from other GGTs. We studied the roles of these residues in the distinct biochemical properties of PnGGT using site-directed mutagenesis. All mutants showed a significant decrease in hydrolysis activity and an increase in transpeptidase activity, suggesting that the aromatic side chains of Trp385, Phe417, and Trp525 were involved in the recognition of acceptor substrates. Abbreviations: ϒ-glutamyl peptide, theanine, X-ray crystallography.

Hyperstabilization of Tetrameric Bacillus sp. TB-90 Urate Oxidase by Introducing Disulfide Bonds through Structural Plasticity.

Bacillus sp. TB-90 urate oxidase (BTUO) is one of the most thermostable homotetrameric enzymes. We previously reported [Hibi, T., et al. (2014) Biochemistry 53, 3879-3888] that specific binding of a sulfate anion induced thermostabilization of the enzyme, because the bound sulfate formed a salt bridge with two Arg298 residues, which stabilized the packing between two ß-barrel dimers. To extensively characterize the sulfate-binding site, Arg298 was substituted with cysteine by site-directed mutagenesis. This substitution markedly increased the protein melting temperature by ∼ 20 °C compared with that of the wild-type enzyme, which was canceled by reduction with dithiothreitol. Calorimetric analysis of the thermal denaturation suggested that the hyperstabilization resulted from suppression of the dissociation of the tetramer into the two homodimers. The crystal structure of R298C at 2.05 Å resolution revealed distinct disulfide bond formation between the symmetrically related subunits via Cys298, although the Cß distance between Arg298 residues of the wild-type enzyme (5.4 Å apart) was too large to predict stable formation of an engineered disulfide cross-link. Disulfide bonding was associated with local disordering of interface loop II (residues 277-300), which suggested that the structural plasticity of the loop allowed hyperstabilization by disulfide formation. Another conformational change in the C-terminal region led to intersubunit hydrogen bonding between Arg7 and Asp312, which probably promoted mutant thermostability. Knowledge of the disulfide linkage of flexible loops at the subunit interface will help in the development of new strategies for enhancing the thermostabilization of multimeric proteins.

Structural and functional analysis of the yeast N-acetyltransferase Mpr1 involved in oxidative stress tolerance via proline metabolism.

Mpr1 (sigma1278b gene for proline-analog resistance 1), which was originally isolated as N-acetyltransferase detoxifying the proline analog L-azetidine-2-carboxylate, protects yeast cells from various oxidative stresses. Mpr1 mediates the L-proline and L-arginine metabolism by acetylating L-Δ(1)-pyrroline-5-carboxylate, leading to the L-arginine-dependent production of nitric oxide, which confers oxidative stress tolerance. Mpr1 belongs to the Gcn5-related N-acetyltransferase (GNAT) superfamily, but exhibits poor sequence homology with the GNAT enzymes and unique substrate specificity. Here, we present the X-ray crystal structure of Mpr1 and its complex with the substrate cis-4-hydroxy-L-proline at 1.9 and 2.3 Å resolution, respectively. Mpr1 is folded into α/ß-structure with eight-stranded mixed ß-sheets and six α-helices. The substrate binds to Asn135 and the backbone amide of Asn172 and Leu173, and the predicted acetyl-CoA-binding site is located near the backbone amide of Phe138 and the side chain of Asn178. Alanine substitution of Asn178, which can interact with the sulfur of acetyl-CoA, caused a large reduction in the apparent kcat value. The replacement of Asn135 led to a remarkable increase in the apparent Km value. These results indicate that Asn178 and Asn135 play an important role in catalysis and substrate recognition, respectively. Such a catalytic mechanism has not been reported in the GNAT proteins. Importantly, the amino acid substitutions in these residues increased the L-Δ(1)-pyrroline-5-carboxylate level in yeast cells exposed to heat stress, indicating that these residues are also crucial for its physiological functions. These studies provide some benefits of Mpr1 applications, such as the breeding of industrial yeasts and the development of antifungal drugs.

Nanoparticle-assisted laser desorption/ionization using sinapic acid-modified iron oxide nanoparticles for mass spectrometry analysis.

Iron oxide-based nanoparticles (NP) were covalently modified with sinapic acid (SA) through a condensation reaction to assist the ionization of both large and small molecules. The morphology of SA-modified NPs (SA-NP) was characterized by transmission electron microscopy (TEM), and the modification of the NP surface with SA was confirmed using ultraviolet (UV) and infrared (IR) spectroscopy. The number of SA molecules was estimated to be 6 per NP. SA-NP-assisted laser desorption/ionization was carried out on small molecules, such as pesticides and plant hormones, and large molecules, such as peptides and proteins. A peptide fragment from degraded proteins was detected more efficiently compared with conventional methods.

Colorimetric determination of fructose for the high-throughput microtiter plate assay of glucose isomerase.

A colorimetric method for the reducing monosaccharide determination is optimized for the assay of glucose isomerase, which converts glucose (Glc) to fructose (Fru). Test solution was mixed with 20-fold volume of the 50 mM Na2SiO3, 600 mM Na2MoO4, and 0.95 M HCl aqueous solution (pH 4.5), in which a yellow molybdosilicate species was formed. The mixture was kept at 70 °C for 30 min. Test solution containing 10 mM level Fru gave a remarkable blue reaction mixture, in which the Mo(VI) species was reduced by Fru to form a blue molybdosilicate species. The blueness increased with the Fru concentration. Glc cannot render the reaction mixture blue as strong as Fru. Thus, the colorimetric method can be used advantageously for the determination of 10 mM level Fru in the Glc isomerase reaction mixture, even in the presence of 100 mM level Glc, and has been applied successfully to the microtiter plate assay of the enzyme.

Intersubunit salt bridges with a sulfate anion control subunit dissociation and thermal stabilization of Bacillus sp. TB-90 urate oxidase.

The optimal activity of Bacillus sp. TB-90 urate oxidase (BTUO) is 45 °C, but this enzyme is one of the most thermostable urate oxidases. A marked increase (>10 °C) in its thermal stability is induced by high concentrations (0.8­1.2 M) of sodium sulfate. Calorimetric measurements and size exclusion chromatographic analyses suggested that sulfate-induced thermal stabilization is related to the binding of a sulfate anion that repressed the dissociation of BTUO tetramers into dimers. To determine the sulfate binding site, the crystal structure was determined at 1.75 Å resolution. The bound sulfate anion was found at the subunit interface of the symmetrical related subunits and formed a salt bridge with two Arg298 residues in the flexible loop that is involved in subunit assembly. Site-directed mutagenesis of Arg298 to Glu was used to extensively characterize the sulfate binding site at the subunit interface. The network of charged hydrogen bonds via the bound sulfate is suggested to contribute significantly to the thermal stabilization of both subunit dimers and the tetrameric assembly of BTUO. Knowledge of the mechanism of salt-induced stabilization will help to develop new strategies for enhancing protein thermal stabilization.

Overexpression, purification, and characterization of Paenibacillus cell surface-expressed chitinase ChiW with two catalytic domains.

Paenibacillus sp. strain FPU-7 produces several different chitinases and effectively hydrolyzes robust chitin. Among the P. FPU-7 chitinases, ChiW, a novel monomeric chitinase with a molecular mass of 150 kDa, is expressed as a cell surface molecule. Here, we report that active ChiW lacking the anchoring domains in the N-terminus was successfully overproduced in Escherichia coli and purified to homogeneity. The two catalytic domains at the C-terminal region were classified as typical glycoside hydrolase family 18 chitinases, whereas the N-terminal region showed no sequence similarity to other known proteins. The vacuum-ultraviolet circular dichroism spectrum of the enzyme strongly suggested the presence of a ß-stranded-rich structure in the N-terminus. Its biochemical properties were also characterized. Various insoluble chitins were hydrolyzed to N,N'-diacetyl-D-chitobiose as the final product. Based on amino acid sequence similarities and site-directed mutagenesis, Glu691 and Glu1177 in the two GH-18 domains were identified as catalytic residues.

Cooperative degradation of chitin by extracellular and cell surface-expressed chitinases from Paenibacillus sp. strain FPU-7.

Chitin, a major component of fungal cell walls and invertebrate cuticles, is an exceedingly abundant polysaccharide, ranking next to cellulose. Industrial demand for chitin and its degradation products as raw materials for fine chemical products is increasing. A bacterium with high chitin-decomposing activity, Paenibacillus sp. strain FPU-7, was isolated from soil by using a screening medium containing α-chitin powder. Although FPU-7 secreted several extracellular chitinases and thoroughly digested the powder, the extracellular fluid alone broke them down incompletely. Based on expression cloning and phylogenetic analysis, at least seven family 18 chitinase genes were found in the FPU-7 genome. Interestingly, the product of only one gene (chiW) was identified as possessing three S-layer homology (SLH) domains and two glycosyl hydrolase family 18 catalytic domains. Since SLH domains are known to function as anchors to the Gram-positive bacterial cell surface, ChiW was suggested to be a novel multimodular surface-expressed enzyme and to play an important role in the complete degradation of chitin. Indeed, the ChiW protein was localized on the cell surface. Each of the seven chitinase genes (chiA to chiF and chiW) was cloned and expressed in Escherichia coli cells for biochemical characterization of their products. In particular, ChiE and ChiW showed high activity for insoluble chitin. The high chitinolytic activity of strain FPU-7 and the chitinases may be useful for environmentally friendly processing of chitin in the manufacture of food and/or medicine.

A selective hybrid fluorescent sensor for fructose detection based on a phenylboronic acid and BODIPY-based hydrophobicity probe.

Fructose is widely used in the food industry. However, it may be involved in diseases by generating harmful advanced glycation end-products. We have designed and synthesized a novel fluorescent probe for fructose detection by combining a phenylboronic acid group with a BODIPY-based hydrophobicity probe. This probe showed a linear fluorescence response to d-fructose concentration in the range of 100-1000 µM, with a detection limit of 32 µM, which is advantageous for the simple and sensitive determination of fructose.

Identification of quasi-stable water molecules near the Thr73-Lys13 catalytic diad of Bacillus sp. TB-90 urate oxidase by X-ray crystallography with controlled humidity.

Urate oxidases (UOs) catalyze the cofactor-independent oxidation of uric acid, and an extensive water network in the active site has been suggested to play an essential role in the catalysis. For our present analysis of the structure and function of the water network, the crystal qualities of Bacillus sp. TB-90 urate oxidase were improved by controlled dehydration using the humid air and glue-coating method. After the dehydration, the P21212 crystals were transformed into the I222 space group, leading to an extension of the maximum resolution to 1.42 Å. The dehydration of the crystals revealed a significant change in the five-water-molecules' binding mode in the vicinity of the catalytic diad, indicating that these molecules are quasi-stable. The pH profile analysis of log(kcat) gave two pKa values: pKa1 at 6.07 ± 0.07 and pKa2 at 7.98 ± 0.13. The site-directed mutagenesis of K13, T73 and N276 involved in the formation of the active-site water network revealed that the activities of these mutant variants were significantly reduced. These structural and kinetic data suggest that the five quasi-stable water molecules play an essential role in the catalysis of the cofactor-independent urate oxidation by reducing the energy penalty for the substrate-binding or an on-off switching for the proton-relay rectification.

Structural characterization of two solute-binding proteins for N,N'-diacetylchitobiose/N,N',N''-triacetylchitotoriose of the gram-positive bacterium, Paenibacillus sp. str. FPU-7.

The chitinolytic bacterium Paenibacillus sp. str. FPU-7 efficiently degrades chitin into oligosaccharides such as N-acetyl-D-glucosamine (GlcNAc) and disaccharides (GlcNAc)2 through multiple secretory chitinases. Transport of these oligosaccharides by P. str. FPU-7 has not yet been clarified. In this study, we identified nagB1, predicted to encode a sugar solute-binding protein (SBP), which is a component of the ABC transport system. However, the genes next to nagB1 were predicted to encode two-component regulatory system proteins rather than transmembrane domains (TMDs). We also identified nagB2, which is highly homologous to nagB1. Adjacent to nagB2, two genes were predicted to encode TMDs. Binding experiments of the recombinant NagB1 and NagB2 to several oligosaccharides using differential scanning fluorimetry and surface plasmon resonance confirmed that both proteins are SBPs of (GlcNAc)2 and (GlcNAc)3. We determined their crystal structures complexed with and without chitin oligosaccharides at a resolution of 1.2 to 2.0 Å. The structures shared typical SBP structural folds and were classified as subcluster D-I. Large domain motions were observed in the structures, suggesting that they were induced by ligand binding via the "Venus flytrap" mechanism. These structures also revealed chitin oligosaccharide recognition mechanisms. In conclusion, our study provides insight into the recognition and transport of chitin oligosaccharides in bacteria.

Functional analysis of α-1,3-glucanase domain structure from Streptomyces thermodiastaticus HF3-3.

α-1,3-Glucanase from Streptomyces thermodiastaticus HF3-3 (Agl-ST) has been classified in the glycoside hydrolase (GH) family 87. Agl-ST is a multi-modular domain consisting of an N-terminal ß-sandwich domain (ß-SW), a catalytic domain, an uncharacterized domain (UC), and a C-terminal discoidin domain (DS). Although Agl-ST did not hydrolyze α-1,4-glycosidic bonds, its amino acid sequence is more similar to GH87 mycodextranase than to α-1,3-glucanase. It might be categorized into a new subfamily of GH87. In this study, we investigated the function of the domains. Several fusion proteins of domains with green fluorescence protein (GFP) were constructed to clarify the function of each domain. The results showed that ß-SW and DS domains played a role in binding α-1,3-glucan and enhancing the hydrolysis of α-1,3-glucan. The binding domains, ß-SW and DS, also showed binding activity toward xylan, although it was lower than that for α-1,3-glucan. The combination of ß-SW and DS domains demonstrated high binding and hydrolysis activities of Agl-ST toward α-1,3-glucan, whereas the catalytic domain showed only a catalytic function. The binding domains also achieved effective binding and hydrolysis of α-1,3-glucan in the cell wall complex of Schizophyllum commune.

Molecular cloning and characterization of γ-glutamyltranspeptidase from Pseudomonas nitroreducens IFO12694.

γ-glutamyltranspeptidase from Pseudomonas nitroreducens IFO12694 (PnGGT) exhibited higher hydrolytic activity than transfer activity, as compared with other γ-glutamyltranspeptidases (GGTs). PnGGT showed little activity towards most of L-amino acids and towards glycyl-glycine, which is often used as a standard γ-glutamyl accepter in GGT transfer reactions. The preferred substrates for PnGGT as a γ-glutamyl accepter were amines such as methylamine, ethylamine, and isopropylamine.

Threonine at position 306 of the KAT1 potassium channel is essential for channel activity and is a target site for ABA-activated SnRK2/OST1/SnRK2.6 protein kinase.

The Arabidopsis thaliana K+ channel KAT1 has been suggested to have a key role in mediating the aperture of stomata pores on the surface of plant leaves. Although the activity of KAT1 is thought to be regulated by phosphorylation, the endogenous pathway and the primary target site for this modification remained unknown. In the present study, we have demonstrated that the C-terminal region of KAT1 acts as a phosphorylation target for the Arabidopsis calcium-independent ABA (abscisic acid)-activated protein kinase SnRK2.6 (Snf1-related protein kinase 2.6). This was confirmed by LC-MS/MS (liquid chromatography tandem MS) analysis, which showed that Thr306 and Thr308 of KAT1 were modified by phosphorylation. The role of these specific residues was examined by single point mutations and measurement of KAT1 channel activities in Xenopus oocyte and yeast systems. Modification of Thr308 had minimal effect on KAT1 activity. On the other hand, modification of Thr306 reduced the K+ transport uptake activity of KAT1 in both systems, indicating that Thr306 is responsible for the functional regulation of KAT1. These results suggest that negative regulation of KAT1 activity, required for stomatal closure, probably occurs by phosphorylation of KAT1 Thr306 by the stress-activated endogenous SnRK2.6 protein kinase.

Structural insights into substrate recognition and catalysis by glycoside hydrolase family 87 α-1,3-glucanase from Paenibacillus glycanilyticus FH11.

The α-1,3-glucanase from Paenibacillus glycanilyticus FH11 (Agl-FH1), a member of the glycoside hydrolase family 87 (GH87), hydrolyzes α-1,3-glucan with an endo-action. GH87 enzymes are known to degrade dental plaque produced by oral pathogenic Streptococcus species. In this study, the kinetic analyses revealed that this enzyme hydrolyzed α-1,3-tetraglucan into glucose and α-1,3-triglucan with ß-configuration at the reducing end by an inverting mechanism. The crystal structures of the catalytic domain (CatAgl-FH1) complexed with or without oligosaccharides at 1.4-2.5 or 1.6 Å resolutions, respectively, are also presented. The initial crystal structure of CatAgl-FH1 was determined by native single-wavelength anomalous diffraction. The catalytic domain was composed of two modules, a ß-sandwich fold module, and a right-handed ß-helix fold module. The structure of the ß-sandwich was similar to those of the carbohydrate-binding module family 35 members. The glycerol or nigerose enzyme complex structures demonstrated that this ß-sandwich fold module is a novel carbohydrate-binding module with the capabilities to bind saccharides and to promote the degradation of polysaccharides. The structures of the inactive mutant in complexes with oligosaccharide showed that at least eight subsites for glucose binding were located in the active cleft of the ß-helix fold and the architecture of the active cleft was suitable for the recognition and hydrolysis of α-1,3-glucan by the inverting mechanism. The structural similarity to GH28 and GH49 enzymes and the results of site-directed mutagenesis indicated that three Asp residues, Asp1045, Asp1068, and Asp1069, are the most likely candidates for the catalytic residues of Agl-FH1. DATABASE: Structural data are available in RCSB Protein Data Bank under the accession numbers 6K0M (CatAgl-FH1), 6K0N (WT/nigerose), 6K0P (D1045A/nigerose), 6K0Q (D1068A/nigerose), 6K0S (D1069A/ nigerose), 6K0U (D1068A/oligo), and 6K0V (D1069A/oligo). ENZYMES: Agl-FH1, α-1,3-glucanase (EC3.2.1.59) from Paenibacillus glycanilyticus FH11.

Crystallization and preliminary crystallographic analysis of N-acetyltransferase Mpr1 from Saccharomyces cerevisiae.

Mpr1 is an enzyme that catalyzes the N-acetylation of the toxic L-azetidine-2-carboxylic acid (AZC). Recently, Mpr1 has been shown to reduce levels of intracellular reactive oxygen species (ROS) under oxidative stress. The natural substrate involved in the ROS elimination in vivo is still unknown. Mpr1 has been purified and crystallized in space groups P1 and P3(1)12. X-ray data were collected to 1.9 A resolution from a trigonal crystal soaked with AZC.