• Journal of Life Science
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• v.16 no.3 s.76
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• pp.375-381
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• 2006

Aldehyde reductase was purified to electrophoretic homogeneity from Saccharomyces cerevisiae, and then enzymatic reduction of substituted carbonyl compounds was carried out by using the purified aldehyde reductase as a biocatalyst. Under preparative scale reaction renditions, the enzymatic reduction proceeded in high chemical yield with excellent chemoselectivity. The enzymatic reduction product was identified by TLC, GC, Mass, NMR and FT-IR. Benzoic acid, an inhibitor of aldehyde reductase, also potently inhibited the reduction of substituded carbonyl compounds. This enzyme exhibited a broad substrate specificity , and can utilize both NADH and NADPH as cofactors. The enzyme was strongly inhibited by benzoic acid and quercetin. The apparent Km for 4-cyanobenzaldehyde and 3-nitrobenzamide were 4.894 mM and 0.305 mM, respectively.

• Applied Chemistry for Engineering
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• v.20 no.4
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• pp.449-452
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• 2009

The absence of Fe, Se, and Mo in a minimal medium prevented the production of hydrogen from the anaerobic culture of Escherichia coli MC4100. Fe, Se, and Mo are known to be cofactors of formate dehydrogenase ($FDH_{II}$) of both E. coli and Enterobacter aerogenes. Hence when these trace elements are absent in the minimal medium, hydrogen production through formate dehydrogenation would be inhibited not only in E. coli but also in E. aerogenes. Hydrogen production by E. aerogenes 413 was delayed when lacking these trace elements. Therefore, it is believed that hydrogen production of E. aerogenes is initiated not by the reoxidation of nicotinamide adenine dinucleotide (NADH) but by formate decarboxylation.

• Proceedings of the Korean Society for Bioinformatics Conference
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• 2005.09a
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• pp.421-426
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• 2005

In this paper, we investigate the nonlinear dynamic behavior of TPP (thiamine pyrophosphate) riboswitches in E. coli (Escherichia coli). TPP riboswitches are highly conserved RNA regulatory elements, embedded within the 5’'untranslated region of three TPP biosynthesis operons. The three operons thiCEFSGH, thiMD, and thiBPQ are involved in the biosynthesis, salvage, and transport of TPP, respectively. TPP riboswitches modulate their expressions in response to changing TPP concentration, without involving protein cofactors. Interestingly, the expression of thiMD is regulated at the translational level, while that of thiCEFSGH at both levels of transcription and translation. We develop a mathematical model of the TPP riboswitch’s regulatory system possessed by thiCEFSGH and thiMD, so as to simulate the time-course experiments of TPP biosynthesis in E. coli. The simulation results are validated against three sets of reported experimental data in order to gain insight into the nature of steady states and the stability of TPP riboswitches, and to explain the biological significance of regulating at level of transcription or translation, or even both. Our findings suggest that in the TPP biosynthesis pathway of E. coli, the biological effect of down-regulating thiCEFSGH operon at the translational level by TPP riboswitch is less prominent than that at the transcriptional level.

• BMB Reports
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• v.29 no.2
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• pp.99-104
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• 1996

Site-directed substitutions were made to change the Ile91 of restriction endonuclease EcoRV to either Val, Ala or Gly to identify the role of Ile91 in recognition and catalysis, since substitution of Ile91 with Leu afforded dramatic effects on the activity and properties of restriction endonuclease EcoRV. These changes alter the size of the hydrophobic side chain at position 91 and thus might have revealed the reason for the altered phenotype of Ile91Leu. However, the properties of Ile91Val and Ile91Ala mutants were much like wild type EcoRV, in both activity and metal ion preference. Ile91Gly had very little activity with either $Mg^{2+}$ or $Mn^{2+}$ as cofactors. To try to understand the unusual $Mn^{2+}$ profile of the Ile91Leu mutant, two double mutants, Ile91Leu;Asp90Asn and Ile91Leu;Glu45Met were created. Both double mutants were seriously disabled by the second amino acid change. Ile91Leu;Glu45Met had some residual activity in the $Mn^{2+}$ reaction buffer, whereas the Ile91Leu;Asp90Asn displayed no detectable activity.

• Journal of the Korean Magnetic Resonance Society
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• v.24 no.3
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• pp.86-90
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• 2020

Iron-sulfur (Fe-S) clusters are one of the most ancient yet essential cofactors mediating various essential biological processes. In prokaryotes, Fe-S clusters are generated via several distinctive biogenesis mechanisms, among which the ISC (Iron-Sulfur Cluster) mechanism plays a house-keeping role to satisfy cellular needs for Fe-S clusters. The Escherichia coli ISC mechanism is maintained by several essential protein factors, whose structural characterization has been of great interest to reveal mechanistic details of the Fe-S cluster biogenesis mechanisms. In particular, nuclear magnetic resonance (NMR) spectroscopic approaches have contributed much to elucidate dynamic features not only in the structural states of the protein components but also in the interaction between them. The present minireview discusses recent advances in elucidating structural features of IscU, the key player in the E. coli ISC mechanism. IscU accommodates exceptional structural flexibility for its versatile activities, for which NMR spectroscopy was particularly successful. We expect that understanding to the structural diversity of IscU provides critical insight to appreciate functional versatility of the Fe-S cluster biogenesis mechanism.

• Archives of Plastic Surgery
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• v.35 no.5
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• pp.491-494
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• 2008

Purpose: When deciding a treatment plan in diabetic foot ulcer patients, predicting a possibility of healing wounds is important since not a few patients have poor general condition to get successful wound healing. This study was planned to find out if a serum collagen level can be used as a predictor for healing wounds in diabetic foot patients. Methods: Fifty-seven patients, who visited our clinic from January to June, 2007 for treatment of diabetic foot ulcers, were included in this study. Serum levels of type I collagen were checked using carboxy terminal type I propeptide kits. Simultaneously serum levels of vitamin C and iron, cofactors of collagen synthesis, were checked. The patients were divided into two groups; a group of successfully healed wounds and the other of unhealed wounds. Serum levels of the parameters were compared between the 2 groups. Results: The serum level of collagen was $197.65{\pm}86.26ng/ml$ in a healed group and $87.91{\pm}28.76ng/ml$ in the unhealed group(p<0.05). The serum iron and vitamin C levels were did not show significant differences. Conclusion: The serum collagen level may predict healing or nonhealing wounds in diabetic foot ulcers.

• Journal of Microbiology and Biotechnology
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• v.20 no.6
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• pp.968-973
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• 2010

In order to study its biochemical properties, the integrase (IN) protein of feline foamy virus (FFV) was overexpressed in Escherichia coli, purified by two-step chromatography, (Talon column and heparin column), and characterized in biochemical aspects. For the three enzymatic reactions of the 3'-processing, strand transfer, and disintegration activities, the $Mn^{2+}$ ion was essentially required as a cofactor. Interestingly, $Co^{2+}$ and $Zn^{2+}$ ions were found to act as effective cofactors, whereas other transition elements such as $Ni^{2+}$, $Cu^{2+}$, $La^{3+}$, $Y^{3+}$, $Cd^{2+}$, $Li^{1+}$, $Ba^{2+}$, $Sr^{2+}$, and $V^{3+}$ were not. Regarding the substrate specificity, FFV IN has low substrate specificities as it cleaved in a significant level prototype foamy virus (PFV) U5 LTR substrate as well as FFV U5 LTR substrate, whereas PFV IN did not. Finally, the 3'-processing activity was observed in high concentrations of several solvents such as CHAPS, glycerol, Tween 20, and Triton X-100, which are generally used for dissolution of chemicals in inhibitor screening. Therefore, in this first report showing its biochemical properties, FFV IN is proposed to have low specificities on the use of cofactor and substrate for enzymatic reaction as compared with other retroviral INs.

• BMB Reports
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• v.41 no.1
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• pp.68-71
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• 2008

Flavones are synthesized from flavanones through the action of flavone synthases (FNSs). There are two FNSs, FNS I and II. FNS I is a soluble dioxygenase present in members of the Apiaceae family and FNS II is a membrane bound cytochrome P450 enzyme that has been identified in numerous plant species. In this study, we cloned OsFNS I-1 from rice by RTPCR, expressed it in E. coli, and purified the recombinant protein. By NMR analysis, we found that OsFNS I-1 converted the flavanone (2S)-naringenin into the flavone, apigenin. Moreover, we found that the cofactors oxoglutarate, $FeSO_4$, ascorbate and catalase are required for this reaction. OsFNS I-1 encodes a flavone synthase I. This is the first type I FNS I found outside of the Apiaceae family.

• Bulletin of the Korean Chemical Society
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• v.26 no.2
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• pp.260-264
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• 2005

The nature of the active site of Tobacco acetohydroxyacid synthase (AHAS) in the substrate- and cofactorbinding was studied by kinetics and fluorescence spectroscopy. The substrate saturation curve does not follow Michaelis-Menten kinetics at different temperatures (7, 21 and 37 ${^{\circ}C}$), pH (6.5, 7.5 and 8.5) and buffers (Tris-HCl and MOPS). The concentration of one half of the maximum velocity ($S_{0.5}$) decreased in the following order: pyruvate $\gt$ ThDP $\approx$$Mg^{+2}$ $\gt$ FAD. However, the catalytic efficiency (K$_{cat}/S_{0.5}$) inversely decreased in the following order; FAD $\gt$ $Mg^{+2}$ $\approx$ThDP $\gt$ pyruvate, indicating that the cofactors by in decreasing order; FAD, $Mg^{+2}$, ThDP, affect the catalysis of AHAS. The dissociation constant ($K_d$) of the intrinsic tryptophan fluorescence decreased with the same tendency of the concentration of one half of the maximum velocity ($S_{0.5}$) decreasing order. This data provides evidence that the substrate and cofactor binding natures of the active site, as well as its activation characteristics, resemble those of other ThDP-dependent enzymes.

• Animal cells and systems
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• v.8 no.1
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• pp.49-55
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• 2004

In vertebrates, multisubunit cofactors regulate gene expression through interacting with cell-type- and gene-specific DNA-binding proteins in a chromatin-selective manner. ADD1/SREBP1c regulates fatty acid metabolism and insulin-dependent gene expression through binding to SRE and E-box motif with dual DNA binding specificity. Although its transcriptional and post-translational regulation has been extensively studied, its regulation by interacting proteins is not well understood. To identify cellular proteins that associate with nuclear form of ADD1/SEBP1c, we employed the GST pull-down system with Hela cell nuclei extract. In this study, we demonstrated that Ku proteins interact specifically with ADD1/SREP1c protein. GST pull-down combined with peptide sequencing analysis revealed that Ku80 binds to ADD1/SREBP1c in vitro. Additionally, western blot analysis showed that Ku70, a heterodimerizing partner of Ku80, also associates with ADD1/SREBP1c. Furthermore, co-transfection of Ku70/Ku80 with ADD1/SREBP1c enhanced the transcriptional activity of ADD1/SREBP1c. Taken together, these results suggest that the Ku proteins might be involved in the lipogenic and/or adipogenic gene expression through interacting with ADD1/SREBP1c.