• Journal of Life Science
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• v.21 no.1
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• pp.119-126
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• 2011

There is a growing recognition of the significance of $H_2S$ as a biological signaling molecule involved in vascular and nervous system functions. In mammals, two enzymes in the transsulfuration pathway, cystathionine ${\beta}$-synthase (CBS) and cystathionine ${\gamma}$-lyase (CGL), are believed to be chiefly responsible for $H_2S$ biogenesis. Genetic inborn error of CGL leads to human genetic disease, cystathioninuria, by accumulating cystathionine in the body. This disease is secondarily associated with a wide range of diseases including diabetes insipidus and Down's syndrome. Although the human CGL (hCGL) overexpression is essential for the investigation of its function, structure, reaction specificity, substrate specificity, and protein-protein interactions, there is no clear report concerning optimum overexpression conditions. In this study, we report a detailed analysis of the overexpression conditions of the hCGL using a bacterial system. Maximum overexpression was obtained in conditions of low culture temperature after inducer addition, performing low aeration during overexpression, and using a low concentration inducer (0.1 mM, IPTG) for induction. Expressed hCGL was purified by His-tag affinity column chromatography and confirmed by Western blot using hCGL antibody and enzyme activity analysis. We also report that the His tag with TEV site attached protein exhibits 76% activity for ${\alpha}-{\gamma}$ elimination reaction with L-cystathionine and 88% for ${\alpha}-{\beta}$ elimination reaction with L-cysteine compared to those of wild type hCGL, respectively. His tag with TEV site attached protein also exhibits a 420 nm absorption maximum, which is attributed to the binding cofactor, pyridoxal 5'-phosphate (PLP).

• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.36-36
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• 2017

Out of twenty common protein amino acids, there are many kinds of non protein amino acids (NPAAs) that exist as secondary metabolites and exert ecological functions in plants. Mimosine (Mim), one of those NPAAs derived from L. leucocephala acts as an iron chelator and reversely block mammalian cell cycle at G1/S phases. Cysteine (Cys) is decisive for protein and glutathione that acts as an indispensable sulfur grantor for methionine and many other sulfur-containing secondary products. Cys biosynthesis includes consecutive two steps using two enzymes-serine acetyl transferase (SAT) and O-acetylserine (thiol)lyase (OASTL) and appeared in plant cytosol, chloroplast, and mitochondria. In the first step, the acetylation of the ${\beta}$-hydroxyl of L-serine by acetyl-CoA in the existence of SAT and finally, OASTL triggers ${\alpha}$, ${\beta}$-elimination of acetate from OAS and bind $H_2S$ to catalyze the synthesis of Cys. Mimosine synthase, one of the isozymes of the OASTLs, is able to synthesize Mim with 3-hydroxy-4-pyridone (3H4P) instead of $H_2S$ for Cys in the last step. Thus, the aim of this study was to clone and characterize the cytosolic (Cy) OASTL gene from L. leucocephala, express the recombinant OASTL in Escherichia coli, purify it, do enzyme kinetic analysis, perform docking dynamics simulation analysis between the receptor and the ligands and compare its performance between Cys and Mim synthesis. Cy-OASTL was obtained through both directional degenerate primers corresponding to conserved amino acid region among plant Cys synthase family and the purified protein was 34.3KDa. After cleaving the GST-tag, Cy-OASTL was observed to form mimosine with 3H4P and OAS. The optimum Cys and Mim reaction pH and temperature were 7.5 and $40^{\circ}C$, and 8.0 and $35^{\circ}C$ respectively. Michaelis constant (Km) values of OAS from Cys were higher than the OAS from Mim. Inter fragment interaction energy (IFIE) of substrate OAS-Cy-OASTL complex model showed that Lys, Thr81, Thr77 and Gln150 demonstrated higher attraction force for Cys but 3H4P-mimosine synthase-OAS intermediate complex showed that Gly230, Tyr227, Ala231, Gly228 and Gly232 might provide higher attraction energy for the Mim. It may be concluded that Cy-OASTL demonstrates a dual role in biosynthesis both Cys and Mim and extending the knowledge on the biochemical regulatory mechanism of mimosine and cysteine.

• Korean Journal of Materials Research
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• v.21 no.7
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• pp.357-363
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• 2011

In this paper, high temperature oxidation behavior of newly developed alloys, Ti-6Al-4Fe and Ti-6Al-1Fe, is examined. To understand the effect of Fe on the air oxidation behavior of the Ti-Al-Fe alloy system, thermal oxidation tests are carried out at $700^{\circ}C$ and $800^{\circ}C$ for 96 hours. Ti-6Al-4V alloy is also prepared and tested under the same conditions for comparison with the developed alloys. The oxidation resistance of the Ti-Al-Fe alloy system is superior to that of Ti-6Al-4V alloy. Ti-6Al-4V shows the worst oxidation resistance for all test conditions. This is not a result of the addition of Fe, but rather it is due to the elimination of V, which has deleterious effects on high temperature oxidation. The oxidation of the Ti-Al-Fe alloy system follows the parabolic rate law. At $700^{\circ}C$, Fe addition does not have a noticeable influence on the amount of weight gain of all specimens. However, at $800^{\circ}C$, Ti-6Al-4Fe alloy shows remarkable degradation compared to Ti-6Al-1Fe and Ti-6Al. It is discovered that the formation of $Al_2O_3$, a diffusion resistance layer, is remarkably hindered by a relative decrease of the ${\alpha}$ volume fraction. This is because Fe addition increases the volume fraction of ${\beta}$ phase within the Ti-6Al-xFe alloy system. Activities of Al, Ti, and Fe with respect to the formation of oxide layers are calculated and analyzed to explore the oxidation mechanism.