Biochemical Studies on the Aspartate Aminotransferase from Deinococcus radiodurans R1
Date Issued
2005
Date
2005
Author(s)
Hsu, Yu-Ting
DOI
zh-TW
Abstract
Deinococcus radiodurans R1 is well-known for its extraordinary resistance against high-dose ionizing radiation, UV radiation and hydrogen peroxide. It is an ideal target for the study of survival strategies adopted by extremophiles under various harsh environmental conditions. This thesis aims at the characterization of an enzyme, aspartate aminotransferase (AspAT, EC 2.6.1.1), from D. radiodurans R1.
Aspartate aminotransferase, also known as glutamate oxaloacetate transaminase (GOT), catalyzed the interconversion between glutamate and aspartate. It is involved in the metabolism of nitrogen-containing biomolecules and is essential for energy metabolism. In D. radiodurans R1 AspAT is encoded by the gene aspC of about 1167 bp. AspAT from D. radiodurans R1 has been cloned, expressed in E. coli, purified and characterized. The expressed recombinant protein was purified to apparent homogeneity by Ni-NTA affinity chromatography. The fusion protein (Maltose-binding Protein) was removed by Factor Xa. The cleavage product was further purified by Ni-NTA column again to yield a homogeneous protein. Its molecular mass was confirmed by mass spectral analysis after desalting by reversed-phase HPLC through a C8 column. The purified protein had a specific activity at 104.6 U/mg. This enzyme, designated as DrAspAT, exhibits remarkable thermal tolerance and radioresistance, as compared with AspAT from porcine cytoplasmic. The radioresistance is dependent on protein concentration： the higher the protein concentration, the less damage caused by X-ray.
The molecular structure of DrAspAT was modeled by using Program O and a template structure of Thermus thermophilus AspAT (1bjw; TtAspAT ). Several catalytically important residues have been indenfied from the modeled strucute. In order to test the modeled structure site-directed mutagenesis experiment was carried out on those important residues. The results showed that all the mutant enzymes lost the enzymatic activity, thus confirming the important roles they play in catalysis.
Sequence alignment and analysis showed that DrAspAT is similar to the AspATs from mesophilic organisms in conformation. However, the comparison result showed evolution diversity between those two kinds of AspATs by the obvious difference in the N-terminal sequence and the residues recognizing the distal carboxylate group of the substrate. Moreover, by advanced analysis for the composition of amino acids, DrAspAT contain higher percentage of proline residues and no cysteine in contrast to other mesophilic counterpars, this preference of DrAspAT may partially account for its remarkable structural stability.
As a result, analysis for biochemical studies on DrAspAT with other data about AspATs from various species could give us some clues about extraordinary radioresistance of D. radiodurans R1 and evolution direction of proteins.
Subjects
天門冬胺酸
轉胺酶
耐輻射
奇異球菌
AspAT
Aminotransferase
radioresistance
Deinococcus radiodurans R1
Type
other
File(s)![Thumbnail Image]()
Loading...
Name
ntu-94-R92b46023-1.pdf
Size
23.31 KB
Format
Adobe PDF
Checksum
(MD5):d614a7226ca98d6eb75ef9b24da8a8d5