Identification, characterization, and cloning of a novel aminoacylase, L-pipecolic acid acylase from Pseudomonas species.

L-Pipecolic acid is utilized as a vital component of specific chemical compounds, such as immunosuppressive drugs, anticancer reagents, and anesthetic reagents. We isolated and characterized a novel L-aminoacylase, N-acetyl-L-pipecolic acid-specific aminoacylase (LpipACY), from Pseudomonas sp. AK2. The subunit molecular mass of LpipACY was 45 kDa and was assumed to be a homooctamer in solution. The enzyme exhibited high substrate specificity toward N-acetyl-L-pipecolic acid and a high activity for N-acetyl-L-pipecolic acid and N-acetyl-L-proline. This enzyme was stable at a high temperature (60°C for 10 min) and under an alkaline pH (6.0-11.5). The N-terminal and internal amino acid sequences of the purified enzyme were STTANTLILRNG and IMASGGV, respectively. These sequences are highly consistent with those of uncharacterized proteins from Pseudomonas species, such as amidohydrolase and peptidase. We also cloned and overexpressed the gene coding LpipACY in Escherichia coli. Moreover, the recombinant LpipACY exhibited properties similar to native enzyme. Our results suggest that LpipACY is a potential enzyme for the enzymatic synthesis of L-pipecolic acid. This study provides the first description of the enzymatic characterization of L-pipecolic acid specific amino acid acylase.

Enzymatic and molecular characterization of α-1,3-glucanase (AglST2) from Streptomyces thermodiastaticus HF3-3 and its relation with α-1,3-glucanase HF65 (AglST1).

Extracellular α-1,3-glucanase HF90 (AglST2), with a sodium dodecyl sulfate (SDS)-PAGE-estimated molecular mass of approximately 91 kDa, was homogenously purified from the culture filtrate of Streptomyces thermodiastaticus HF3-3. AglST2 showed a high homology with mycodextranase in an amino acid sequence and demonstrated specificity with an α-1,3-glycosidic linkage of homo α-1,3-glucan. It has been suggested that AglST2 may be a new type of α-1,3-glucanase. The optimum pH and temperature of AglST2 were pH 5.5 and 60°C, respectively. AglST2 action was significantly stimulated in the presence of 5-20% (w/v) NaCl, and 1 mM metal ions Mn2+ and Co2+. On the other hand, it was inhibited by 1 mM of Ag+, Cu2+, Fe2+ and Ni2+. Regarding the stability properties, AglST2 retained more than 80% of its maximum activity over a pH range of 5.0-7.0 at up to 60°C and in the presence of 0-20% (w/v) NaCl. Based on these results, the properties of AglST2 were comparable with those of AglST1, which had been previously purified and characterized from S. thermodiastaticus HF3-3 previously. The N-terminal amino acid sequence of AglST2 showed a good agreement with that of AglST1, suggesting that AglST1 was generated from AglST2 by proteolysis during cultivation. MALDI-TOF mass analysis suggested that AglST1 might be generated from AglST2 by the proteolytic removal of C-terminus polypeptide (approximately 20 kDa). Our investigation thus revealed the properties of AglST2, such as tolerance against high temperature, salts, and surfactants, which have promising industrial applications.

Ecm33 is a novel factor involved in efficient glucose uptake for nutrition-responsive TORC1 signaling in yeast.

Glucose uptake is crucial for providing both an energy source and a signal that regulates cell proliferation. Therefore, it is important to clarify the mechanisms underlying glucose uptake and its transmission to intracellular signaling pathways. In this study, we searched for a novel regulatory factor involved in glucose-induced signaling by using Saccharomyces cerevisiae as a eukaryotic model. Requirement of the extracellular protein Ecm33 in efficient glucose uptake and full activation of the nutrient-responsive TOR kinase complex 1 (TORC1) signaling pathway is shown. Cells lacking Ecm33 elicit a series of starvation-induced pathways even in the presence of extracellular high glucose concentration. This results in delayed cell proliferation, reduced ATP, induction of autophagy, and dephosphorylation of the TORC1 substrates Atg13 and Sch9.

Computational Identification and Characterization of a Promiscuous T-Cell Epitope on the Extracellular Protein 85B of Mycobacterium spp. for Peptide-Based Subunit Vaccine Design.

Tuberculosis (TB) is a reemerging disease that remains as a leading cause of morbidity and mortality in humans. To identify and characterize a T-cell epitope suitable for vaccine design, we have utilized the Vaxign server to assess all antigenic proteins of Mycobacterium spp. recorded to date in the Protegen database. We found that the extracellular protein 85B displayed the most robust antigenicity among the proteins identified. Computational tools for identifying T-cell epitopes predicted an epitope, 181-QQFIYAGSLSALLDP-195, that could bind to at least 13 major histocompatibility complexes, revealing the promiscuous nature of the epitope. Molecular docking simulation demonstrated that the epitope could bind to the binding groove of MHC II and MHC I molecules by several hydrogen bonds. Molecular docking analysis further revealed that the epitope had a distinctive binding pattern to all DRB1 and A and B series of MHC molecules and presented almost no polymorphism in its binding site. Moreover, using "Allele Frequency Database," we checked the frequency of HLA alleles in the worldwide population and found a higher frequency of both class I and II HLA alleles in individuals living in TB-endemic regions. Our results indicate that the identified peptide might be a universal candidate to produce an efficient epitope-based vaccine for TB.

The signaling pathways underlying starvation-induced upregulation of α-mannosidase Ams1 in Saccharomyces cerevisiae.

BACKGROUND: Cells have evolved the mechanisms to survive nutritional shortages in the environment. In Saccharomyces cerevisiae, α-mannosidase Ams1 is known to play a role in catabolism of N-linked free oligosaccharides in the cytosol. Although, this enzyme is also known to be transported selectively from the cytosol to the vacuoles by autophagy, the physiological significance of this transport has not been clarified. METHODS: To elucidate the regulatory mechanism of the activity of Ams1, we assessed the enzymatic activity of the cell free extract of the wild-type and various gene disruptants under different nutritional conditions. In addition, the regulation of Ams1 at both transcription and post-translation was examined. RESULTS: The activity of Ams1 was significantly increased upon the depletion of glucose in the medium. Interestingly, the activity of the enzyme was also stimulated by nitrogen starvation. Our data showed that the activity of Ams1 is regulated by the stress responsive transcriptional factors Msn2/4 through the protein kinase A and the target of rapamycin complex 1 pathways. In addition, Ams1 is post-translationally activated by Pep4-dependent processing in the vacuoles. CONCLUSION: Yeast cells monitor extracellular nutrients to regulate mannoside catabolism via the cellular signaling pathway. GENERAL SIGNIFICANCE: This study revealed that intracellular Ams1 activity is exquisitely upregulated in response to nutrient starvation by induced expression as well as by Pep4-dependent enhanced activity in the vacuoles. The signaling molecules responsible for regulation of Ams1 were also clarified.

Effective treatment for suppression of acrylamide formation in fried potato chips using L-asparaginase from Bacillus subtilis.

It has been reported that acrylamide, a potential carcinogen, is formed from the reaction of L-asparagine (L-Asn) and reducing sugars contained in foods during heating processes and free asparagine is a limiting factor for acrylamide formation. It has been reported that potato products such as potato chips, which are made through heating processes, contain high levels of acrylamide. To decrease the amount of L-Asn in potatoes using L-asparaginase, effective treatment conditions of sliced potatoes with the enzyme have been investigated. By treating sliced potatoes with Bacillus subtilis L-asparaginase II (BAsnase; 4 U/g potato), appriximately 40 % of L-Asn in the sliced potatoes was converted into L-aspartic acid (L-Asp). To make this enzyme more effective, prior to enzymatic treatment, sliced potatoes were freeze-thawed, dried at 90 °C for 20 min, and vacuum treated for 10 min under decompressed condition, resulting in the hydrolysis of approximately 90 % of L-Asn to L-Asp. The acrylamide content of BAsnase-treated fried potato chips decreased to below 20 % of that of BAsnase-untreated fried potato chips. Treatment conditions examined in this study were found to be effective to suppress the formation of acrylamide in fried potato chips.

Crystal structure of salt-tolerant glutaminase from Micrococcus luteus K-3 in the presence and absence of its product L-glutamate and its activator Tris.

Glutaminase from Micrococcus luteus K-3 [Micrococcus glutaminase (Mglu); 456 amino acid residues (aa); 48 kDa] is a salt-tolerant enzyme. Our previous study determined the structure of its major 42-kDa fragment. Here, using new crystallization conditions, we determined the structures of the intact enzyme in the presence and absence of its product L-glutamate and its activator Tris, which activates the enzyme by sixfold. With the exception of a 'lid' part (26-29 aa) and a few other short stretches, the structures were all very similar over the entire polypeptide chain. However, the presence of the ligands significantly reduced the length of the disordered regions: 41 aa in the unliganded structure (N), 21 aa for L-glutamate (G), 8 aa for Tris (T) and 6 aa for both L-glutamate and Tris (TG). L-glutamate was identified in both the G and TG structures, whereas Tris was only identified in the TG structure. Comparison of the glutamate-binding site between Mglu and salt-labile glutaminase (YbgJ) from Bacillus subtilis showed significantly smaller structural changes of the protein part in Mglu. A comparison of the substrate-binding pocket of Mglu, which is highly specific for L-glutamine, with that of Erwinia carotovora asparaginase, which has substrates other than L-glutamine, shows that Mglu has a larger substrate-binding pocket that prevents the binding of L-asparagine with proper interactions.

Characterization of theanine-forming enzyme from Methylovorus mays no. 9 in respect to utilization of theanine production.

For development of theanine production from glutamic acid and ethylamine by coupling yeast sugar fermentation as an ATP-regenerating system, several strains were selected from among about 200 methylamine- and/or methanol-assimilating bacteria depending on the theanine-forming activity of their permeated cells. The amount of theanine formed by the cells of the selected strains was much larger than that by the cells of Escherichia coli AD494 (DE3) expressing Pseudomonas taetrolens Y-30 glutamine synthetase (GS), which has been found to be a usable enzyme for theanine production. A GS-like enzyme responsible for the theanine-forming reaction was obtained from an obligate methylotroph isolate, Methylovorus mays No. 9. The enzyme was induced by methylamine in the culture medium. A molecular mass of 410-470 kDa was obtained by gel filtration of the enzyme, and 51 kDa by SDS-PAGE analysis. The enzyme showed high activity toward methylamine rather than ammonia, which indicates that it is similar to known gamma-glutamylmethylamide synthetase. The isolated enzyme also had high reactivity to ethylamine in a neutral pH range, and formed theanine from glutamic acid and ethylamine in a reaction mixture containing a yeast sugar fermentation system for ATP-regeneration.

Theanine production by coupled fermentation with energy transfer employing Pseudomonas taetrolens Y-30 glutamine synthetase and baker's yeast cells.

Theanine was formed from glutamic acid and ethylamine by coupling the reaction of glutamine synthetase (GS) of Pseudomonas taetrolens Y-30 with sugar fermentation of baker's yeast cells as an ATP-regeneration system. Theanine formation was stimulated by the addition of Mn2+ to the mixture for the coupling. The addition of Mg2+ was less effective. In a mixture containing a larger amount of yeast cells with a fixed level of GS, glucose (the energy source) was consumed rapidly, resulting in a decrease in the final yield of theanine. On the other hand, an increase in GS amounts increased theanine formation in a mixture with a fixed amount of yeast cells. High concentrations of ethylamine enhanced theanine formation whereas inhibited yeast fermentation of sugar and the two contrary effects of ethylamine caused a high yield of theanine based on glucose consumed. In an improved reaction mixture containing 200 mM sodium glutamate, 1,200 mM ethylamine, 300 mM glucose, 50 mM potassium phosphate buffer (pH 7.0), 5 mM MnCl2, 5 mM AMP, 100 units/ml GS, and 60 mg/ml yeast cells, approximately 170 mM theanine was formed in 48 h.

Purification and characterization of glutamine synthetase of Pseudomonas taetrolens Y-30: an enzyme usable for production of theanine by coupling with the alcoholic fermentation system of baker's yeast.

Concentrated cell-extract of Pseudomonas taetrolens Y-30, isolated as a methylamine-assimilating organism, formed gamma-glutamylethylamide (theanine) from glutamic acid and ethylamine in a mixture containing the alcoholic fermentation system of baker's yeast for ATP-regeneration. Glutamine synthetase (GS), probably responsible for theanine formation, was isolated from the extract of the organism grown on a medium containing 1% methylamine, 1% glycerol, 0.5% yeast extract, and 0.2% polypepton as carbon and nitrogen sources. The molecular mass was estimated to be 660 kDa by gel filtration and 55 kDa by SDS-polyacrylamide gel electrophoresis, suggesting that Ps. taetrolens Y-30 GS consists of 12 identical subunits. The enzyme required Mg2+ or Mn2+ for its activity. Under the standard reaction condition for glutamine formation (pH 8.0 with 30 mM Mg2+), GS showed 7% and 1% reactivity toward methylamine and ethylamine respectively of that to ammonia. Reactivity to the alkylamines varied with optimum pH of the reaction in response to divalent cation in the mixture: pH 11.0 was the optimum for the Mg2+ -dependent reaction with ethylamine, and pH 8.5 was the optimum for the Mn2+ -dependent reaction. In a mixture of an optimum reaction condition with 1000 mM ethylamine (at pH 8.5 with 3 mM Mn2+), reactivity increased up to 7% of the reactivity to ammonia in the standard reaction condition. The isolated GS formed theanine in the mixture with the yeast fermentation system.