• BMB Reports
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• v.38 no.1
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• pp.58-64
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• 2005

Myo-inositol monophosphate phosphatase (IMPP) is a key enzyme in the phosphoinositide cell-signaling system. This study found that incubating the IMPP from a porcine brain with pyridoxal-5'-phosphate (PLP) resulted in a time-dependent enzymatic inactivation. Spectral evidence showed that the inactivation proceeds via the formation of a Schiff's base with the amino groups of the enzyme. After the sodium borohydride reduction of the inactivated enzyme, it was observed that 1.8 mol phosphopyridoxyl residues per mole of the enzyme dimer were incorporated. The substrate, myo-inositol-1-phosphate, protected the enzyme against inactivation by PLP. After tryptic digestion of the enzyme modified with PLP, a radioactive peptide absorbing at 210 nm was isolated by reverse-phase HPLC. Amino acid sequencing of the peptide identified a portion of the PLP-binding site as being the region containing the sequence L-Q-V-S-Q-Q-E-D-I-T-X, where X indicates that phenylthiohydantoin amino acid could not be assigned. However, the result of amino acid composition of the peptide indicated that the missing residue could be designated as a phosphopyridoxyl lysine. This suggests that the catalytic function of IMPP is modulated by the binding of PLP to a specific lysyl residue at or near its substrate-binding site of the protein.

• Journal of Microbiology and Biotechnology
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• v.26 no.4
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• pp.730-738
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• 2016

est19 is a gene from Bacillus sp. K91 that encodes a new esterase. A comparison of the amino acid sequence showed that Est19 has typical Ser-Gly-Asn-His (SGNH) family motifs and could be grouped into the SGNH hydrolase family. The Est19 protein was functionally cloned, and expressed and purified from Escherichia coli BL21(DE3). The enzyme activity was optimal at 60℃ and pH 9.0, and displayed esterase activity towards esters with short-chain acyl esters (C₂-C₆). A structural model of Est19 was constructed using phospholipase A1 from Streptomyces albidoflavus NA297 as a template. The structure showed an α/β-hydrolase fold and indicated the presence of the typical catalytic triad Ser49-Asp227-His230, which were further investigated by site-directed mutagenesis. To the best of our knowledge, Est19 is a new member of the SGNH hydrolase family identified from thermophiles, which may be applicable in the industrial production of semisynthetic β-lactam antibiotics after modification.

• Applied Chemistry for Engineering
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• v.29 no.2
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• pp.185-190
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• 2018

The dimer of bicyclo [2.2.1] hepta-2,5-diene (norbornadiene) can be used as a high-energy-density fuel. The purpose of this study is to investigate the effect of Co loading on the acid properties of HY zeolite catalyst and the catalytic activity in norbornadiene dimerization. When the cobalt was loaded on the HY zeolite catalyst, the amount of acid sites did not change, but the acid strength weakened. This can be attributed to the decrease in $Br{\ddot{o}}nsted$ acid site and the increase in Lewis acid site. The norbornadiene conversion and yield of norbornadiene dimer over the Co/HY catalyst showed higher than those over the HY zeolite catalyst. The higher activity of the Co/HY catalyst can be ascribed to the higher amount of Lewis acid sites over the Co/HY catalyst. Density and calorific values of the norbornadiene dimer prepared by using the Co/HY catalyst agreed well with the known values in the literature. It was confirmed that the norbornadiene dimer prepared in this study can be used as a high-energy-density fuel.

• Applied Biological Chemistry
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• v.40 no.3
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• pp.189-195
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• 1997

The structural basis of Iysine specificity of Achromobacter protease I (API) was investigated by means of site-directed mutagenesis. The precursor protein in which Glu190, one of the two candidates for determining Iysine specificity, was substituted by glutamine, aspartic acid or leucine was processed autocatalytically to attaln full pretense activity with lysine specificity. The substitution of the other candidate, Asp225, for asparagine or leucine produced no mature active forms of pro-API. The precursor protein of the mutant D225E slowly matured autocatalytically. The lysylendopeptidase activity of the mature D225E was 0.25% of that of native API, and this reduced activity is mainly due to a decrease in the affinity of the enzyme for lysine. These results suggest that Asp225 plays a critical rol in restricted substrate specificity as a lysylendopeptidase. However, D225E exhibited no measurable activity for synthetic ornithine substrate. Since the hydroxyl group of Ser194 in this mutant retained essentially the same reactivity to DFP or PMSF as that in native API, it can be noted that a methylene unit longer side chain of residue 225 is not compensated by a methylene unit shorter side chain at subsite P1 in the bound substrate.

• Journal of Life Science
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• v.17 no.1 s.81
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• pp.6-11
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• 2007

[ $\beta$ ]-Fructofuranosidases, a family 32 of glycoside hydrolases (GH32), share three conserved domains including the W(L/M)(C/N)DP(Q/N), FRDPK, and ECP(D/G) motifs. The functional role of the conserved acidic residues within three domains of levan fructotransferase, one of the $\beta-fructofuranosidases$, from Microbacterium sp. AL-210 was studied by site-directed mutagenesis. Each mutant was overexpressed in E. coli BL21(DE3) and purified by using Hi-Trap chelating affinity chromatography and fast performance liquid chromatography. Substitution of Asp-63 by Ala, Asp-195 by Asn, and Glu-245 by Ala and Asp decreased the enzyme activity by approximately 100-fold compared to the wild-type enzyme. This result indicates that three acidic residues Asp-63, Asp-195, and Glu-245 play a major role in catalysis. Since the three acidic residues are present in a conserved position in inulinase, levanase, levanfructotransferase, and invertase, they are likely to have a common functional role as nucleophile, transition state stabilizer, and general acid in $\beta-fructofuranosidases$.

• Journal of Plant Biotechnology
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• v.33 no.1
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• pp.1-9
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• 2006

Peroxiredoxins (Prxs) are ubiquitously distributed and play important functions in diverse cellular signaling systems. The proteins are largely classified into three groups, such as typical 2-Cys Prx, atypical 2-Cys Prx, and 1-Cys Prx, that are distinguished by their catalytic mechanisms and number of Cys residues. From the three classes of Prxs, the typical 2-Cys Prx containing the two-conserved Cys residues at its N-terminus and C-terminus catalyzes $H_2O_2$ with the use of thioredoxin (Trx) as an electron donor. During the catalytic cycle, the N-terminal Cys residue undergoes a peroxide-dependent oxidation to sulfenic acid, which can be further oxidized to sulfinic acid at the presence of high concentrations of $H_2O_2$ and a Trx system containing Trx, Trx reductase, and NADPH. The sulfinic acid form of 2-Cys Prx is reduced by the action of sulfiredoxin which requires ATP as an energy source. Under the strong oxidative or heat shock stress conditions, 2-Cys Prx in eukaryotes rapidly switches its protein structure from low-molecular-weight species to high-molecular-weight protein structures. In accordance with its structural changes, the protein concomitantly triggers functional switching from a peroxidase to a molecular chaperone, which can protect its substrate denaturation from external stress. In addition to its N-terminal active site, the C-terminal domain including 'YF-motif' of 2-Cys Prx plays a critical role in the structural changes. Therefore, the C-terminal truncated 2-Cys Prxs are not able to regulate their protein structures and highly resistant to $H_2O_2$-dependent hyperoxidation, suggesting that the reaction is guided by the peroxidatic Cys residue. Based on the results, it may be concluded that the peroxidatic Cys of 2-Cys Prx acts as an '$H_2O_2$-sensor' in the cells. The oxidative stress-dependent regulation of 2-Cys Prx provides a means of defense systems in cells to adapt stress conditions by activating intracellular defense signaling pathways. Particularly, 2-Cys Prxs in plants are localized in chloroplasts with a dynamic protein structure. The protein undergoes conformational changes again oxidative stress. Depending on a redox-potential of the chloroplasts, the plant 2-Cys Prx forms super-molecular weight protein structures, which attach to the thylakoid membranes in a reversible manner.

• Journal of Life Science
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• v.23 no.1
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• pp.15-23
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• 2013

AprE51 from Bacillus amyloliquefaciens CH51 is a 27 kDa subtilisin-like protease with fibrinolytic activity. AprE51-6 showing increased catalytic activity was produced previously. To enhance the thermostability of AprE51-6, 2 residues, Gly-166 and Asn-218 based on B. subtilis subtilisin E were mutated by site-directed mutagenesis. The results of the mutational analysis showed that substitution of arginine for Gly-166 (AprE51-7) increased the fibrinolytic activity 1.8-fold. An N218S mutant (AprE51-8) also increased the fibrinolytic activity up to 4.5-fold in a fibrin plate assay. Purified AprE51-7 and AprE51-8 mutants had a 1.9- and a 2.5-fold higher $k_{cat}$, respectively, and a 2.1-1.9-fold lower $K_m$, respectively. This resulted in a 3.8- and a 4.7-fold increase in catalytic efficiency ($k_{cat}/K_m$), respectively, relative to that of wild-type AprE51. AprE51-8 had a broader pH range than AprE51-6 and nattokinase, especially at an alkaline pH value. In addition, AprE51-8 showed higher thermostability than AprE51-6 at $60^{\circ}C$. The half-lives of AprE51-7 and AprE51-8 at $50^{\circ}C$ were 21.5 and 27.3 min, respectively, which are 2.0 and 2.6 times longer, respectively, than that of the wild-type AprE51.

• Journal of Korean Society of Environmental Engineers
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• v.22 no.4
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• pp.609-617
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• 2000

This study is aimed at investigating an effect of HCl gas on selective reduction of NOx over a CuHM and $V_2O_5-WO_3/TiO_2$ catalyst. SCR process is the most effective method to remove NOx, but catalyst can be deactivated by the acidic gas such as HCl gas which is also included in flue gas from the incinerator. In dry condition of flue gas, the CuHM catalyst treated by HCl gas has shown higher NO removal activity than the fresh catalyst. The activity of the catalyst can be restored by treating at $500^{\circ}C$. On the contrary. $V_2O_5-WO_3/TiO_2$ catalyst is obviously deactivated by HCl and the deactivation increases in proportion to the concentration of HCl gas. The deactivated catalyst is not restored to it's original activity by heat treatment for regeneration. In wet flue gas stream, the CuHM catalyst has shown lower activity than fresh catalyst and $V_2O_5-WO_3/TiO_2$ catalyst was severely deactivated by HCl treatment. The activity loss of catalysts are mainly due to the decrease of Br$\ddot{o}$nsted acid site on the catalyst surface by $NH_3$ TPD. The change of BET surface area of CuHM catalyst after the reaction isn't observed but $V_2O_5-WO_3/TiO_2$ catalyst is observed. The amount of $Cu^{{+}{+}}$ and $V_2O_5$ is decreased after the reaction. From these results, it is expected that CuHM catalyst should be better than $V_2O_5-WO_3/TiO_2$ catalyst for its application to the incineration of flue gas.

• Transactions of the Korean Society of Mechanical Engineers B
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• v.34 no.2
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• pp.165-171
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• 2010

The Homogeneous Charge Compression Ignition (HCCI) engine concept allows for both NOx and particulate matter to be reduced simultaneously, and it is a promising way to meet the next environmental challenges. Unfortunately, HCCI combustion often increases CO and HC emissions. The development of oxidation catalyst (OC) requires high conversion efficiency for CO and HC at low temperature. Conventional oxidation catalyst technologies may not be able to convert these emissions because of the saturation of active catalytic sites. The OC used in this study was 600 cpsi cordierite. Three kinds of OC with different amounts of Pt and Pd were used. The influence of the space velocity (SV), $H_2O$ and $O_2$ concentration was also studied. All types of OCs were found to have over 90% CO conversion efficiencies at $170^{\circ}C$. When in the presence of water vapor, CO conversion was increased, but $C_3H_8$ conversion was decreased. The performance of the OC was not influenced by initial the HC concentration. The 2Pt/Pd catalyst was better in terms of thermal aging than the Pt-only catalyst. The $LOT_{50}$ of both fresh and aged OC was increased with increasing SV and with the presence of $H_2O$.

• BMB Reports
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• v.42 no.12
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• pp.840-845
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• 2009

Mitogen- and stress-activated protein kinase (MSK1) palys a crucial role in the regulation of transcription downstream of extracellular-signal-regulated kinase1/2 (ERK1/2) and mitogen-activated protein kinase p38. MSK1 can be phosphorylated and activated in cells by both ERK1/2 and p38$\alpha$. In this study, Casein Kinase 2 (CK2) was identified as a binding and regulatory partner for MSK1. Using the yeast two-hybrid system, MSK1 was found to interact with the CK2$\beta$ regulatory subunit of CK2. Interactions between MSK1 and the CK2$\alpha$ catalytic subunit and CK2$\beta$ subunit were demonstrated in vitro and in vivo. We further found that CK2$\alpha$ can only interact with the C-terminal kinase domain of MSK1. Using site-directed mutagenesis assay and mass spectrometry, we identified five sites in the MSK1 C-terminus that could be phosphorylated by CK2 in vitro: Ser757, Ser758, Ser759, Ser760 and Thr793. Of these, Ser757, Ser759, Ser760 and Thr793 were previously unknown.