Tatsuki Kashiwagi

The novel acidophilic structure of the killer toxin from halotolerant yeast demonstrates remarkable folding similarity with a fungal killer toxin

BACKGROUND Several strains of yeasts and fungi produce proteinous substances, termed killer toxins, which kill sensitive strains. The SMK toxin, secreted by the halotolerant yeast Pichia farinosa KK1 strain, uniquely exhibits its maximum killer activity under conditions of acidic pH and high salt concentration. The toxin is composed of two distinct subunits, alpha and beta, which tightly interact with each other under acidic conditions. However, they are easily dissociated under neutral conditions and lose the killer activity. The three-dimensional structure of the SMK toxin will provide a better understanding of the mechanism of toxicity of this protein and the cause of its unique pH-dependent stability. RESULTS Two crystal structures of the SMK toxin have been determined at 1.8 A resolution in different ionic strength conditions. The two subunits, alpha and beta, are jointly folded into an ellipsoidal, single domain structure belonging to the alpha/beta-sandwich family. The folding topology of the SMK toxin is essentially the same as that of the fungal killer toxin, KP4. This shared topology contains two left-handed split betaalphabeta motifs, which are rare in the other proteins. Many acidic residues are clustered at the bottom of the SMK toxin molecule. Some of the carboxyl sidechains interact with each other through hydrogen bonds. The ionic strength difference induces no evident structural change of the SMK toxin except that, in the high ionic strength crystal, a number of sulfate ions are electrostatically bound near the basic residues which are also locally distributed at the bottom of the toxin molecule. CONCLUSIONS The two killer toxins, SMK and KP4, share a unique folding topology which contains a rare structural motif. This observation may suggest that these toxins are evolutionally and/or functionally related. The pH-dependent stability of the SMK toxin is a result of the intensive interactions between the carboxyl groups. This finding is important for protein engineering, for instance, towards stabilization of the toxin molecule in a broader pH range. The present crystallographic study revealed that the structure of the SMK toxin itself is hardly affected by the ionic strength, implying that a high salt concentration affects the sensitivity of the cell against the toxin.

Enhancement of transglutaminase activity by NMR identification of its flexible residues affecting the active site

Incorporation of inter‐ or intramolecular covalent cross‐links into food proteins with microbial transglutaminase (MTG) improves the physical and textural properties of many food proteins, such as tofu, boiled fish paste, and sausage. By using nuclear magnetic resonance, we have shown that the residues exhibiting relatively high flexibility in MTG are localized in the N‐terminal region; however, the N‐terminal region influences the microenvironment of the active site. These results suggest that the N‐terminal region is not of primary importance for the global fold, but influences the substrate binding. Therefore, in order to increase the transglutaminase activity, the N‐terminal residues were chosen as candidates for site‐directed replacement and deletion. We obtained several mutants with higher activity, del1–2, del1–3, and S2R. We propose a strategy for enzyme engineering targeted toward flexible regions involved in the enzymatic activity. In addition, we also briefly describe how the number of glutamine residues in a substrate protein can be increased by mixing more than two kinds of TGases with different substrate specificities.

Curculin Exhibits Sweet-tasting and Taste-modifying Activities through Its Distinct Molecular Surfaces.

Curculin isolated from Curculigo latifolia, a plant grown in Malaysia, has an intriguing property of modifying sour taste into sweet taste. In addition to this taste-modifying activity, curculin itself elicits a sweet taste. Although these activities have been attributed to the heterodimeric isoform and not homodimers of curculin, the underlying mechanisms for the dual action of this protein have been largely unknown. To identify critical sites for these activities, we performed a mutational and structural study of recombinant curculin. Based on the comparison of crystal structures of curculin homo- and heterodimers, a series of mutants was designed and subjected to tasting assays. Mapping of amino acid residues on the three-dimensional structure according to their mutational effects revealed that the curculin heterodimer exhibits sweet-tasting and taste-modifying activities through its partially overlapping but distinct molecular surfaces. These findings suggest that the two activities of the curculin heterodimer are expressed through its two different modes of interactions with the T1R2-T1R3 heterodimeric sweet taste receptor.

Crystal structure of Bifidobacterium Longum phosphoketolase; key enzyme for glucose metabolism in Bifidobacterium

MINT‐7985878: PKT (uniprotkb:Q6R2Q7) and PKT (uniprotkb:Q6R2Q7) bind (MI:0407) by X‐ray crystallography (MI:0114)

An electrospray-ionization mass spectrometry analysis of the pH-dependent dissociation and denaturation processes of a heterodimeric protein

Electrospray ionization mass spectrometry (ESI-MS) was applied to the analysis of the dissociation and denaturation processes of a heterodimeric yeast killer toxin SMKT. The two distinct subunits of SMKT noncovalently associate under acidic conditions, but become dissociated and denatured under neutral and basic conditions. In order to understand the unique pH-dependent denaturation mechanism of this protein, a pH titration was performed by utilizing ESI-MS. The molecular ions of the heterodimer which possesses the highly ordered structure, were mainly observed below pH 4.6. However, the two subunits immediately dissociated at this pH. The spectra measured with various settings of the mass spectrometer indirectly demonstrated that the pH-dependent dissociation occurs in the liquid phase. The current result as well as the three-dimensional structure of SMKT suggest that the deprotonation of a specific carboxyl group triggers a cooperative dissociation process of this protein. In conclusion, the pH titration of a protein by ESI-MS is particularly effective, when the unfolding process or the biological function of the protein is related to the interaction with other molecules.

Crystallization and preliminary X-ray analysis of the periplasmic receptor (PotF) of the putrescine transport system in Escherichia coli

The primary receptor (PotF) of the putrescine transport system in E. coli has been crystallized by the hanging-drop vapor-diffusion technique. The crystals belong to the space group P21212 with unit-cell dimensions a = 269.4, b = 82.33 and c = 93.74 A. The crystals diffract beyond 2.2 A with a rotating-anode X-ray source. A complete data set from the native crystals has been collected and processed at 2.3 A resolution. Two heavy-atom derivatives have been prepared from the same Pt compound at 293 and 277 K. The difference Patterson maps revealed completely different major heavy-atom sites between these two derivatives.

Preparation and Characterization of Four Stereoisomers of Monatin.

Monatin is a naturally occurring, sweet amino acid comprising four stereoisomers due to its two asymmetric centers at C2 and C4. However, the characteristics of each stereoisomer have not yet been fully investigated. To obtain a sufficient amount of racemic monatin for optical resolution, a synthetic method was developed by modifying a possible biosynthetic pathway, i.e., a cross-aldol reaction and subsequent transamination. The key intermediate, 4-hydroxy-4-(3-indolylmethyl)-2-ketoglutaric acid, was obtained via the cross-aldol reaction of pyruvic acid and indole-3-pyruvic acid. Subsequently, the carbonyl group was converted to a hydroxyimino group through reaction with hydroxylamine and then to an amino group via hydrogenation to produce monatin. Next, the racemic monatin was divided into mixtures of two pairs of enantiomers through recrystallization. Finally, both enantiomers of the N-carbobenzoxy-γ-lactone derivatives of monatin were separated by preparative HPLC and deprotected. It was found that all optically pure stereoisomers exhibited a sweet taste. The isomer that displayed the most intense sweetness was the (2R,4R)-isomer, as determined by single crystal X-ray structure analysis of the monatin potassium salt, whereas the least sweet isomer was the (2S,4S)-isomer, which demonstrated a far lower sweetness than was previously reported.

Interaction Analysis of FABP4 Inhibitors by X-ray Crystallography and Fragment Molecular Orbital Analysis

X-ray crystal structural determination of FABP4 in complex with four inhibitors revealed the complex binding modes, and the resulting observations led to improvement of the inhibitory potency of FABP4 inhibitors. However, the detailed structure-activity relationship (SAR) could not be explained from these structural observations. For a more detailed understanding of the interactions between FABP4 and inhibitors, fragment molecular orbital analyses were performed. These analyses revealed that the total interfragment interaction energies of FABP4 and each inhibitor correlated with the ranking of the K i value for the four inhibitors. Furthermore, interactions between each inhibitor and amino acid residues in FABP4 were identified. The oxygen atom of Lys58 in FABP4 was found to be very important for strong interactions with FABP4. These results might provide useful information for the development of novel potent FABP4 inhibitors.

Crystallization and preliminary X‐ray diffraction studies of a novel killer toxin from a halotolerant yeast Pichia farinosa

A killer toxin from a halotolerant yeast, Pichia farinosa strain KK1, was crystallized at high- and low-salt concentrations. Crystals from the high-salt solution belonged to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell dimensions of a = b = 81.10, c = 118.46 A. The low-salt solution provided crystals that belonged to the same space group, with nearly same cell dimensions. Preliminary diffraction studies showed that the intensity distributions are significantly different between the two crystals. Both types of crystals contained either two or three molecules per asymmetric unit. They diffracted X-rays beyond 2.0 A resolution and were stable to X-ray irradiation.

In-situ and real-time growth observation of high-quality protein crystals under quasi-microgravity on earth.

Precise protein structure determination provides significant information on life science research, although high-quality crystals are not easily obtained. We developed a system for producing high-quality protein crystals with high throughput. Using this system, gravity-controlled crystallization are made possible by a magnetic microgravity environment. In addition, in-situ and real-time observation and time-lapse imaging of crystal growth are feasible for over 200 solution samples independently. In this paper, we also report results of crystallization experiments for two protein samples. Crystals grown in the system exhibited magnetic orientation and showed higher and more homogeneous quality compared with the control crystals. The structural analysis reveals that making use of the magnetic microgravity during the crystallization process helps us to build a well-refined protein structure model, which has no significant structural differences with a control structure. Therefore, the system contributes to improvement in efficiency of structural analysis for “difficult” proteins, such as membrane proteins and supermolecular complexes.