Kohki Ishikawa

X-ray structures of a novel acid phosphatase from Escherichia blattae and its complex with the transition-state analog molybdate.

The structure of Escherichia blattae non‐specific acid phosphatase (EB‐NSAP) has been determined at 1.9 Å resolution with a bound sulfate marking the phosphate‐binding site. The enzyme is a 150 kDa homohexamer. EB‐NSAP shares a conserved sequence motif not only with several lipid phosphatases and the mammalian glucose‐6‐phosphatases, but also with the vanadium‐containing chloroperoxidase (CPO) of Curvularia inaequalis. Comparison of the crystal structures of EB‐NSAP and CPO reveals striking similarity in the active site structures. In addition, the topology of the EB‐NSAP core shows considerable similarity to the fold of the active site containing part of the monomeric 67 kDa CPO, despite the lack of further sequence identity. These two enzymes are apparently related by divergent evolution. We have also determined the crystal structure of EB‐NSAP complexed with the transition‐state analog molybdate. Structural comparison of the native enzyme and the enzyme–molybdate complex reveals that the side‐chain of His150, a putative catalytic residue, moves toward the molybdate so that it forms a hydrogen bond with the metal oxyanion when the molybdenum forms a covalent bond with NE2 of His189.

Stabilization of Escherichia coli ribonuclease HI by cavity-filling mutations within a hydrophobic core.

The crystal structure of Escherichia coli ribonuclease HI has a cavity near Val-74 within the protein core. In order to fill the cavity space, we constructed two mutant proteins, V74L and V74I, in which Val-74 was replaced with either Leu or Ile, respectively. The mutant proteins are stabilized, as revealed by a 2.1-3.7 degrees C increase in the Tm values, as compared to the wild-type protein at pH values of 3.0 and 5.5. The mutant protein V74A, in which Val-74 is replaced with Ala, was also constructed to analyze the reverse effect. The stability of V74A decreases by 7.6 degrees C at pH 3.0 and 12.7 degrees C at pH 5.5 in Tm as compared to those values for the wild-type protein. None of the three mutations significantly affect the enzymatic activity. The crystal structures of V74L and V74I, determined at 1.8-A resolution, are almost identical to that of the wild-type protein, except for the mutation site. In the two mutant proteins, calculation by the Voronoi procedure shows that the cavity volumes around the individual mutation sites are remarkably reduced as compared to that in the wild-type protein. These results indicate that the introduction of a methylene group into the cavity, without causing steric clash, contributes to an increase in the hydrophobic interaction within the protein core and thereby enhances protein stability. We also discuss the role of the Leu side chain, which can assume many different local conformations on a helix without sacrificing thermostability.

Crystal Structure of Red Sea Bream Transglutaminase

The crystal structure of the tissue-type transglutaminase from red sea bream liver (fish-derived transglutaminase, FTG) has been determined at 2.5-Å resolution using the molecular replacement method, based on the crystal structure of human blood coagulation factor XIII, which is a transglutaminase zymogen. The model contains 666 residues of a total of 695 residues, 382 water molecules, and 1 sulfate ion. FTG consists of four domains, and its overall and active site structures are similar to those of human factor XIII. However, significant structural differences are observed in both the acyl donor and acyl acceptor binding sites, which account for the difference in substrate preferences. The active site of the enzyme is inaccessible to the solvent, because the catalytic Cys-272 hydrogen-bonds to Tyr-515, which is thought to be displaced upon acyl donor binding to FTG. It is postulated that the binding of an inappropriate substrate to FTG would lead to inactivation of the enzyme because of the formation of a new disulfide bridge between Cys-272 and the adjacent Cys-333 immediately after the displacement of Tyr-515. Considering the mutational studies previously reported on the tissue-type transglutaminases, we propose that Cys-333 and Tyr-515 are important in strictly controlling the enzymatic activity of FTG.

Highly diastereoselective aldol reaction of fluoroalkyl aryl ketones with methyl isocyanoacetate catalyzed by silver(I)/triethylamine

Abstract Transition metal-catalyzed aldol reaction of aryl fluoroalkyl ketones with methyl isocyanoacetate was found to proceed with high diastereoselectivity that is in sharp contrast to the very poor diastereoselectivity in the similar reaction of nonfluorinated ketones. The superiority of AgClO4/NEt3 as a homogeneous catalyst (1 mol %) for these reactions was disclosed.

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.

Improving the Pyrophosphate-inosine Phosphotransferase Activity of Escherichia blattae Acid Phosphatase by Sequential Site-directed Mutagenesis

Escherichia blattae acid phosphatase/phosphotransferase (EB-AP/PTase) exhibits C-5′-position selective pyrophosphate-nucleoside phosphotransferase activity in addition to its intrinsic phosphatase. Improvement of its phosphotransferase activity was investigated by sequential site-directed mutagenesis. By comparing the primary structures of higher 5′-inosinic acid (5′-IMP) productivity and lower 5′-IMP productivity acid phosphatase/phosphotransferase, candidate residues of substitution were selected. Then a total of 11 amino acid substitutions were made with sequential substitutions. As the number of substituted amino acid residues increased, the 5′-IMP productivity of the mutant enzyme increased, and the activity of the 11 mutant phosphotransferases of EB-AP/PTase reached the same level as that of Morganella morganii AP/PTase. This result shows that Leu63, Ala65, Glu66, Asn69, Ser71, Asp116, Thr135, and Glu136, whose relevance was not directly established by structural analysis alone, also plays an important role in the phosphotransferase activity of EB-AP/PTase.

The structure of brazzein, a sweet-tasting protein from the wild African plant Pentadiplandra brazzeana

Brazzein is the smallest sweet-tasting protein and was isolated from the wild African plant Pentadiplandra brazzeana. The brazzein molecule consists of 54 amino-acid residues and four disulfide bonds. Here, the first crystal structure of brazzein is reported at 1.8 Å resolution and is compared with previously reported solution structures. Despite the overall structural similarity, there are several remarkable differences between the crystal and solution structures both in their backbone folds and side-chain conformations. Firstly, there is an additional α-helix in the crystal structure. Secondly, the atomic r.m.s.d.s between the corresponding C(α)-atom pairs are as large as 2.0-2.2 Å between the crystal and solution structures. Thirdly, the crystal structure exhibits a molecular shape that is similar but not identical to the solution structures. The crystal structure of brazzein reported here will provide additional information and further insights into the intermolecular interaction of brazzein with the 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)

Crystallization and preliminary X-ray analysis of brazzein, a new sweet protein

Brazzein is a sweet protein isolated from a wild African plant Pentadiplandra brazzeana. Brazzein is the smallest (molecular mass = 6473 Da) and the most water-soluble protein sweetener discovered so far and is highly thermostable. Crystals were grown by vapor diffusion using sodium sulfate as a precipitant. They belong to the tetragonal space group I4(1)22 with unit-cell parameters a = b = 61.4, c = 59.6 A and with one molecule in the asymmetric unit. The crystals diffract to 1.8 A resolution using synchrotron radiation.

Rapid Identification of Ligand-Binding Sites by Using an Assignment-Free NMR Approach

In this study, we developed an assignment-free approach for rapid identification of ligand-binding sites in target proteins by using NMR. With a sophisticated cell-free stable isotope-labeling procedure that introduces (15)N- or (13)C-labels to specific atoms of target proteins, this approach requires only a single series of ligand titrations with labeled targets. Using titration data, ligand-binding sites in the target protein can be identified without time-consuming assignment procedures. We demonstrated the feasibility of this approach by using structurally well-characterized interactions between mitogen-activated protein (MAP) kinase p38α and its inhibitor 2-amino-3-benzyloxypyridine. Furthermore, we confirmed the recently proposed fatty acid binding to p38α and confirmed the fatty acid-binding site in the MAP kinase insert region.