• BMB Reports
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• v.41 no.7
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• pp.516-522
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• 2008

The glycation of BSA leads to protein/peptide modifications that result in the formation of AGEs. AGEs react with the amino groups of N-terminal amino acid residues, particularly arginine and lysine residues. Enhanced AGE formation exists in the blood and tissues of diabetics, as well as in aging and other disorders. The Identification of AGEs is of great importance. Mass spectrometry has been applied to identify and structurally elucidate AGEs. Here, we report on the identification of AGE-peptides and AGE precursors based on relative mass changes as a result of specific AGE formation. HPLC-ESIMS, ESI-MS/MS, and the Mascot database were used. The relative mass changes due to the specific type of AGE formation were added to the identified peptides followed by a manual search of the glycated samples, which resulted in the identification of seven peptides for the formation of five AGEs, namely CML, pyrraline, imidazolone A, imidazolone B, and AFGP. Four glycated peptides (FPK, ECCDKPLLEK, IETMR, and HLVDEPQNLIK) were identified in the formation of AGE-precursors.

• Molecules and Cells
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• v.45 no.7
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• pp.495-501
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• 2022

Leucine dehydrogenase (LDH, EC 1.4.1.9) catalyzes the reversible deamination of branched-chain L-amino acids to their corresponding keto acids using NAD⁺ as a cofactor. LDH generally adopts an octameric structure with D4 symmetry, generating a molecular mass of approximately 400 kDa. Here, the crystal structure of the LDH from Pseudomonas aeruginosa (Pa-LDH) was determined at 2.5 Å resolution. Interestingly, the crystal structure shows that the enzyme exists as a dimer with C2 symmetry in a crystal lattice. The dimeric structure was also observed in solution using multiangle light scattering coupled with size-exclusion chromatography. The enzyme assay revealed that the specific activity was maximal at 60℃ and pH 8.5. The kinetic parameters for three different amino acid and the cofactor (NAD⁺) were determined. The crystal structure represents that the subunit has more compact structure than homologs' structure. In addition, the crystal structure along with sequence alignments indicates a set of non-conserved arginine residues which are important in stability. Subsequent mutation analysis for those residues revealed that the enzyme activity reduced to one third of the wild type. These results provide structural and biochemical insights for its future studies on its application for industrial purposes.

• Korean Journal of Microbiology
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• v.48 no.3
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• pp.180-186
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• 2012

Pseudomonas aeruginosa, a Gram-negative bacterium, is an ubiquitous and opportunistic human pathogen, which expresses many virulence factors through quorum sensing (QS) regulation. QscR, one of the QS signal receptors of P. aeruginosa, has unique features that make it possible to distinguish QscR from other QS receptors. In the present study, we focused on amino acid residues responsible for such a broad signal specificity of QscR. Thus we constructed mutant QscRs: $QscR_{T72I}$, $QscR_{R132M}$, and $QscR_{T140I}$ by substituting $72^{nd}$ threonine, $132^{nd}$ arginine, and $140^{th}$ threonine residues with isoleucine, methionine, and isoleucine, respectively by site-directed mutagenesis. When we examined the activity of these mutant QscRs, $QscR_{R132M}$ failed to respond to N-3-oxododecanoyl homoserine lactone (3OC12-HSL), but $QscR_{T72I}$ and $QscR_{T140I}$ remained the ability to respond to 3OC12-HSL despite much reduction of the sensitivity. When we treated a variety of acyl-HSLs with different structure, $QscR_{T72I}$ and $QscR_{T140I}$ showed better responsiveness to N-decanoyl HSL (C10-HSL) or N-dodecanoyl HSL (C12-HSL) that has no oxo-moiety at $3^{rd}$ carbon of acyl group than to 3OC12-HSL, and $QscR_{R132M}$ showed no responsiveness to any acyl-HSLs tested here. In addition, $QscR_{T72I}$ and $QscR_{T140I}$ were inhibited by 5f, a QscR inhibitor as similarly as wild type QscR was. These results suggest that while the $130^{th}$ arginine is crucial in both activity and acyl-HSL binding of QscR, the $72^{nd}$ and $140^{th}$ threonines are important in the activity, but they are little responsible for the discrimination of acyl-HSLs or competitive inhibitor.

• Journal of Microbiology and Biotechnology
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• v.15 no.5
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• pp.1125-1129
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• 2005

Cloning of a 6.6-kb BamHI digested chromosomal DNA from S. griseus IFO13350 revealed the presence of an additional gene encoding a novel trypsin-like enzyme, named SprU. The SprU protein shows a high homology ($79\%$ identity, $88\%$ similarity) with the SGT protease, which has been reported as a bacterial trypsin in the same strain. The amino acid sequence deduced from the nucleotide sequence of the sprU gene suggests that SprU is produced as a precursor consisting of an amino-terminal presequence (29 amino acid residues), prosequence (4 residues), and mature trypsin consisting of 222 amino acids with a molecular weight of 22.94 kDa and a calculated pI of 4.13. The serine, histidine, and aspartic acid residues composing the catalytic triad of typical serine proteases are also well conserved. When the trypsin activity of the SprU was spectrophotometrically measured by the enzymatic hydrolysis of the artificial chromogenic substrate, N-${alpha}$-benzoyl-DL-arginine-p-nitroanilide, the S. lividans transformant with pWHM3-U gave 3 times higher activity than that of control. When the same recombinant plasmid was introduced into S. griseus, however, the gene dosage effect was not so significant, as in the cases of other genes encoding serine proteases, such as sprA, sprB, and sprD. Although two trypsins, SprU and SGT, have a high degree of homology, the pI values, the gene dosage effect in S. griseus, and the gene arrangement adjacent to the two genes are very different, suggesting that the biochemical and biological function of the SprU might be quite different from that of the SGT.

• BMB Reports
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• v.46 no.11
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• pp.555-560
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• 2013

Acrolein is the most reactive aldehydic product of lipid peroxidation and is found to be elevated in the brain when oxidative stress is high. The effects of acrolein on the structure and function of human Cu,Zn-superoxide dismutase (SOD) were examined. When Cu,Zn-SOD was incubated with acrolein, the covalent crosslinking of the protein was increased, and the loss of enzymatic activity was increased in a dose-dependent manner. Reactive oxygen species (ROS) scavengers and copper chelators inhibited the acrolein-mediated Cu,Zn-SOD modification and the formation of carbonyl compound. The present study shows that ROS may play a critical role in acrolein-induced Cu,Zn-SOD modification and inactivation. When Cu,Zn-SOD that has been exposed to acrolein was subsequently analyzed by amino acid analysis, serine, histidine, arginine, threonine and lysine residues were particularly sensitive. It is suggested that the modification and inactivation of Cu,Zn-SOD by acrolein could be produced by more oxidative cell environments.

• YAKHAK HOEJI
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• v.31 no.3
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• pp.173-181
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• 1987

Protein methylase I has been partially purified from hog pancreas with a 11% yeild. The final preparation is completely free of any other protein-specific methyltransferases and endogenous substrate proteins. The enzyme has an optimum pH of 7.2 and the approximate molecular weight is above 800 thousands dalton. The Km values for S-adenosyl-L-methionine and histone type II-A are 1.32$\times$10$^{-5}$M. The Ki value for S-adenosyl-L-homocysteine is 1.52$\times$10$^{-6}$M. The effect of enyzme concentration on the activity showed a slight sigmoidal curve suggesting the involvement of certain cofactors. Even though the purified enzyme showed two bands on polyacrylamide gel electrophoresis, the enzyme is highly specific for the arginine residues of protein and specifically, highly specific for histone, suggesting histonespecific protein methylase I.

• Journal of Microbiology and Biotechnology
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• v.20 no.3
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• pp.532-541
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• 2010

Peroxidase-like activity of Vitreoscilla hemoglobin (VHb) has been recently disclosed. To maximize such activity, two catalytically conserved residues (histidine and arginine) found in the distal pocket of peroxidases have successfully been introduced into that of the VHb. A 15-fold increase in catalytic constant ($k_{cat}$) was obtained in P54R variant,which was presumably attributable to the lower rigidity and higher hydrophilicity of the distal cavity arising from substitution of proline to arginine. None of the modifications altered the affinity towards either $H_2O_2$ or ABTS substrate. Spectroscopic studies revealed that VHb variants harboring the T29H mutation apparently demonstrated a spectral shift in both ferric and ferrous forms (406-408 to 411 nm, and 432 to 424-425 nm, respectively). All VHb proteins in the ferrous state had a $\lambda_{soret}$ peak at ~419 nm following the carbon monoxide (CO) binding. Expression of the P54R mutant mediated the downregulation of iron superoxide dismutase (FeSOD) as identified by two-dimensional gel electrophoresis (2-DE) and peptide mass fingerprinting (PMF). According to the high peroxidase activity of P54R, it could effectively eliminate autoxidation-derived $H_2O_2$, which is a cause of heme degradation and iron release. This decreased the iron availability and consequently reduced the formation of the $Fe^{2+}$-ferric uptake regulator protein ($Fe^{2+}$-Fur), an inducer of FeSOD expression.

• Journal of Life Science
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• v.21 no.3
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• pp.473-479
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• 2011

Adenylyl cyclase (AC) catalyzes the formation of cyclic AMP (cAMP) from ATP. The cAMP produced by AC serves as a secondary messenger in a variety of signal transduction pathways, and controls various cellular functions in many organisms. ACs can be grouped into six classes based on their primary amino acid sequences. Eukaryotes and mycobacteria contain only members of class III AC. The catalytic cyclase domains of class III AC are active as dimers: mammalian ACs, which are composed of a single polypeptide with two catalytic cyclase domains, form the active site as a result of intramolecular dimerization of the catalytic cyclase domains. In contrast, mycobacterial ACs function as homodimers, since their polypeptides contain a single catalytic cyclase domain. Six amino acids are required for the catalytic activity of class III AC - two aspartate residues, a lysine-aspartate pair and an arginine-asparagine pair. 16 ACs belonging to the class III were identified in Mycobacterium tuberculosis H37Rv, and their characteristics are reviewed.

• Journal of Microbiology and Biotechnology
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• v.28 no.5
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• pp.671-678
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• 2018

Papiliocin, isolated from the swallowtail butterfly (Papilio xuthus), is an antimicrobial peptide with high selectivity against gram-negative bacteria. We previously showed that the N-terminal helix of papiliocin (PapN) plays a key role in the antibacterial and anti-inflammatory activity of papiliocin. In this study, we measured the selectivity of PapN against multidrug-resistant gram-negative bacteria, as well as its anti-inflammatory activity. Interactions between Trp2 of PapN and lipopolysaccharide (LPS), which is a major component of the outer membrane of gram-negative bacteria, were studied using the Trp fluorescence blue shift and quenching in LPS micelles. Furthermore, using circular dichroism, we investigated the interactions between PapN and LPS, showing that LPS plays critical roles in peptide folding. Our results demonstrated that Trp2 in PapN was buried deep in the negatively charged LPS, and Trp2 induced the ${\alpha}$-helical structure of PapN. Importantly, docking studies determined that predominant electrostatic interactions of positively charged arginine residues in PapN with phosphate head groups of LPS were key factors for binding. Similarly, hydrophobic interactions by aromatic residues of PapN with fatty acid chains in LPS were also significant for binding. These results may facilitate the development of peptide antibiotics with anti-inflammatory activity.

• Microbiology and Biotechnology Letters
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• v.30 no.1
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• pp.39-45
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• 2002

It was found that the peptides originated from the hydrolysates of silk fibroin have in vitro immunostimulating effects in murine macrophage RAW264.7 cells. The stimulation effects on nitric oxide (NO) production resulted from treatments of acid or enzymatic hydrolysates were measured. The silk fibroin preparation isolated from cocoon was most efficiently digested by acid hydrolysis. Even though the sole treatment of acid hydrolysate stimulated the NO production in dose-dependent pattern, a part of its activity was found to be caused by the contaminated endotoxin, LPS. When each endotoxin-free hydrolysates obtained by filtering it through an ultrafiltration membrane of molecular weight (MW) cut-off 10,000 to eliminate LPS was used, the peptic hydrolysate with lowest degree of hydrolysis showed the highest activity. The fractions of peptic hydrolysate with MW ranges of 1,000∼10,000, 500∼1,000 and below 500 also showed a higher MW-higher activity correlation. From the analyses of amino acid composition of each hydrolysate, it was found that the contents of arginine, lysine, alanine and glycine residues affected the activity level of hydrolysate. The results of this study showed a possibility of utilizing fibroin as a source for immunostimulating (chemopreventive) functional peptides.