嗜熱菌與抗輻射菌歧鏈胺基酸轉胺酶之研究

Abstract

歧鏈胺基酸轉胺酶 (Branched-Chain Amino Acid Aminotransferase; BCAT) 催化歧鏈胺基酸：白胺酸 (leucine)、異白胺酸 (isoleucine) 和纈胺酸 (valine) 的轉胺作用，結構上，屬於PLP依賴性酵素中的fold typeⅣ，是藉由PLP輔酶的re-face方位催化反應。BCAT 可以用來製造歧鏈胺基酸，例如L-tert-leucine。由於BCAT對於受質沒有專一性，更容易應用於非蛋白質胺基酸的製造。 Deinococcus radiodurans R1最廣泛讓人知的是極端抗游離輻射，DrBCAT (D. radiodurans BCAT) 基因被選殖和表現在大腸桿菌M15宿主細胞，用金屬親和性層析 (Ni-NTA) 及離子交換層析法純化達到均質。利用逆相HPLC去鹽後，以質譜儀測定分子量，結果與理論值完全相符。純化過後的蛋白質有非常高的比活性：150 U/mg，DrBCAT 為雙聚體，它並不耐熱，在65度10分鐘就會減少67%活性。 Thermus thermophilus HB8為一極端嗜熱的菌，它所含的BCAT (TtBCAT) 基因被選殖和表現在大腸桿菌，用金屬親和性層析 (Ni-NTA) 純化到均質。利用逆相HPLC去鹽後，以質譜儀測定分子量，結果與理論值完全相符。所得到的蛋白質比活性跟E.coli BCAT相近，約為5-15 U/mg 。但却比E. coli BCAT還要耐熱，可以在80℃加熱10分鐘還不會改變活性大小，在100℃加熱10分鐘才會失去活性，而TtBCAT與 E. coli和Salmonella的BCAT同為六聚體，有別與其它大部份的BCAT為雙聚體。 利用分子模擬推測DrBCAT的立體構造。由模擬結果得知，DrBCAT的活性中心的胺基酸位置幾乎都與參考結構 (human BCAT) 相同。利用定點突變改變活性中心的重要胺基酸，所得的突變酵素均失去活性。由此可知模擬所得的結果大致是正確的。 經過X-射線照射2.5 kGy劑量，DrBCAT殘留58%的活性，而其它BCAT只剩下44%的活性。此一結果表示DrBCAT具有明顯的耐輻射能力。 這些BCAT具有不同程度的耐熱與耐輻射能力，其分子機制是重要的研究課題。將來經由胺基酸的組成以及蛋白質的立體構造的比較分析，或有可能得到解釋。

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.