Characterization of branched chain amino acid aminotransferase from Thermus thermophilus HB8 and Deinococcus radiodurans R1
Date Issued
2005
Date
2005
Author(s)
Lin, Chih-Hao
DOI
zh-TW
Abstract
Branched-chain aminotransferases (BCATs) catalyze the transamination of branched chain amino acids (BCAAs): leucine, isoleucine, and valine. Structurally, the BCATs belong to the fold type IV class of PLP-dependent enzymes. The catalysis is on the re-face of the PLP cofactor. BCAT can be used to synthesize L-tert-leucine and other branched-chain or unnatural amino acids from their respective keto acids; they have broad applicability in the synthesis of fine chemicals and pharmaceuticals.
Deinococcus radiodurans R1 is best known for its extreme resistance to the lethal effects of ionizing radiation. DrBCAT gene (BCAT from D. radiodurans) was cloned and expressed in E. coli M15. The expressed DrBCAT was purified to apparent homogeneity by affinity chromatography on Ni-NTA column followed by DEAE column chromatography; its molecular mass was confirmed by mass spectral analysis after desalting by reversed-phase HPLC through a C8 column. The purified proteins have high specific activity at 150 U/mg. DrBCAT, a homodimer, was not thermostable; about 67% of activity was lost after incubation at 65℃ for 10 minutes.
Thermus thermophilus HB8, an extremely thermophilic bacterium. Its BCAT (TtBCAT) was cloned and expressed in E. coli M15. The expressed TtBCAT was purified to apparent homogeneity by affinity chromatography on Ni-NTA column; its molecular mass was confirmed by mass spectral analysis after desalting by reversed-phase HPLC through a C8 column. The specific activity of TtBCAT was close to that of E. coli BCAT at 5-15 U/mg. There is no substantial change in its specific activity after incubation at 80℃ for 10 minutes. The activity was lost after incubation at 100℃. It is interesting to note that, as the BCATs from E. coli and Salmonella, TtBCAT also exists as hexamer in solution, whereas the majority of other BCATs are dimeric.
Molecular modeling was used to predict the 3-D structure of DrBCAT, especially the critical amino acid residues at the active site. According to the results, amino acids of active sites are very similar to these of the template structure (human BCAT). Site-directed mutagenesis was used to verify the molecular modeling result.
Different BCATs were irradiated with X-ray at 2.5 kGy to observe the destruction levels of proteins. The results showed that DrBCAT is more radioresistant than other proteins; DrBCATs retain 58% of specific activity, higher than the residual activity of 44%, after X-irradiation.
The rationale behind thermostability and radioresistance of these BCATs could be explained based on the comparison of amino acid compositions and three-dimensional structures of these proteins.
Subjects
歧鏈胺基酸轉胺酶
branched chain amino acid aminotransferase
aminotransferase
Type
other
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