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.

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.

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.

Production of N-acetyl cis-4-hydroxy-L-proline by the yeast N-acetyltransferase Mpr1.

The proline analog cis-4-hydroxy-L-proline (CHOP), which inhibits the biosynthesis of collagen, has been evaluated as an anticancer, antifibrosis, and antihypertension drug. However, its water solubility and low molecular weight limit its therapeutic potential since it is rapidly excreted. In addition, CHOP is considered to be too toxic due primarily to its systematic effects on noncollagen proteins. To promote retention in blood or decrease toxicity, N-acetylation of CHOP might be a novel approach as a prodrug, instead of other approaches such as the conjugation of poly(ethylene glycol-Lys) or the modification of O-acetylation. In this study, we found that N-acetyltransferase Mpr1 that detoxifies the proline analog azetidine-2-carboxylate in Saccharomyces cerevisiae also converts CHOP into N-acetyl CHOP in vitro and in vivo. Escherichia coli BL21(DE3) cells overexpressing Mpr1 showed greater CHOP resistance than those carrying the vector. To increase the productivity of N-acetyl CHOP, the addition of NaCl into the medium that induces osmotic stress accelerates CHOP uptake into E. coli cells. As a result, the amount of N-acetyl CHOP production in Mpr1-overexpressing cells was 3.5-fold higher than that observed in the cells cultured in the absence of NaCl. The highest yield was achieved during the exponential growth phase of cells in the presence of 2% NaCl (52 µmol N-acetyl CHOP per g wet cell weight). Our results provide a promising approach to microbial production of N-acetyl CHOP as a new prodrug.

Application of poly[oxyethylene(dimethylimino)propyl-(dimethylimino)ethylene] as enzyme stabilizer for bilirubin oxidase immobilized electrode.

It has been shown that polyammonium cations comprising quaternary ammonium and hydrophilic groups such as amide and hydroxyl groups stabilize a redox enzyme bilirubin oxidase (BOD). The BOD catalyzes the reaction: 4[Fe(CN)6]4- + 4H+ + O2 --> 4[Fe(CN)6]3- + 2H2O, and has been a promising enzyme for use as a cathode catalyst in biofuel cells. In this study, the stabilizing effect of poly[oxyethylene(dimethylimino)propyl(dimethylimino)ethylene] (PA1) on BOD has been investigated. The sample solution containing BOD and the PA1 salt was kept at a given temperature, and the loss of the enzymatic activity was detected after given stored times. The activity decreased exponentially with stored time so that the first-order rate-constant of inactivation was determined. The inactivation rate-constant lowered with increasing the concentration of the PA1 salt, suggesting that BOD was stabilized by the association with the PA1 cation. The PA1 cation may act like a protective colloid or decrease the local disorder of BOD by its wrapping. A membrane-covered electrode containing BOD, PA1, and [Fe(CN)6](4-/3-) in the internal solution phase was examined in air-saturated aqueous solution. The electrode gave a well-defined current-potential curve with a steady state limiting current due to the PA1-[Fe(CN)6](4-/3-) polyion complex-mediated bioelectrocatalytic current for the reduction of O2. The decreasing of the steady state limiting current became slower in the presence of the PA1 salt, indicating again the stabilizing effect of PA1 cation on BOD.

Crystallization and preliminary crystallographic analysis of bifunctional gamma-glutamylcysteine synthetase-glutatione synthetase from Streptococcus agalactiae.

gamma-Glutamylcysteine synthetase-glutathione synthetase (gammaGCS-GS) is a bifunctional enzyme that catalyzes two consecutive steps of ATP-dependent peptide formation in glutathione biosynthesis. Streptococcus agalactiae gammaGCS-GS is a target for the development of potential therapeutic agents. gammaGCS-GS was crystallized using the sitting-drop vapour-diffusion method. The crystals grew to dimensions of 0.3 x 0.2 x 0.2 mm under reducing conditions with 5 mM TCEP. X-ray data were collected to 2.8 A resolution from a tetragonal crystal that belonged to space group I4(1).

Crystal structure of gamma-glutamylcysteine synthetase: insights into the mechanism of catalysis by a key enzyme for glutathione homeostasis.

Gamma-glutamylcysteine synthetase (gammaGCS), a rate-limiting enzyme in glutathione biosynthesis, plays a central role in glutathione homeostasis and is a target for development of potential therapeutic agents against parasites and cancer. We have determined the crystal structures of Escherichia coli gammaGCS unliganded and complexed with a sulfoximine-based transition-state analog inhibitor at resolutions of 2.5 and 2.1 A, respectively. In the crystal structure of the complex, the bound inhibitor is phosphorylated at the sulfoximido nitrogen and is coordinated to three Mg2+ ions. The cysteine-binding site was identified; it is formed inductively at the transition state. In the unliganded structure, an open space exists around the representative cysteine-binding site and is probably responsible for the competitive binding of glutathione. Upon inhibitor binding, the side chains of Tyr-241 and Tyr-300 turn, forming a hydrogen-bonding triad with the carboxyl group of the inhibitor's cysteine moiety, allowing this moiety to fit tightly into the cysteine-binding site with concomitant accommodation of its side chain into a shallow pocket. This movement is caused by a conformational change of a switch loop (residues 240-249). Based on this crystal structure, the cysteine-binding sites of mammalian and parasitic gammaGCSs were predicted by multiple sequence alignment, although no significant sequence identity exists between the E. coli gammaGCS and its eukaryotic homologues. The identification of this cysteine-binding site provides important information for the rational design of novel gammaGCS inhibitors.

Escherichia coli B gamma-glutamylcysteine synthetase: modification, purification, crystallization and preliminary crystallographic analysis.

Escherichia coli B gamma-glutamylcysteine synthetase (gammaGCS) catalyzes the ATP-dependent coupling of L-Glu and L-Cys to form the glutathione precursor gamma-L-Glu-Cys and is a target for development of potential therapeutic agents. By introducing four point mutations of surface-exposed cysteine residues to serine, the gammaGCS was purified to homogeneity; single crystals have been obtained using the hanging-drop vapour-diffusion method with sodium formate. The gammaGCS crystal diffracted to 2.8 A and belongs to space group R3, with unit-cell parameters a = b = 326.7, c = 103.9 A.