嗜熱性乙醯基轉移酶 SsArd1 之受質專一性辨認與催化機制及其抗熱特性探討

Abstract

蛋白質 N 端乙醯基轉移酶 (Nα-acetyltransferases, Nats) 在細胞中廣泛的影響許多重要的生物功能。近年來研究報導，Nats 也被應用在的臨床藥物的大量製備，例如Ｎ端乙醯化thymosin α1 and thymosin β4，使其轉換成活性態並且於臨床上使用。依據受質前兩個胺基酸組成的不同，使Nats 蛋白質結構有著多元特性的呈現。然而，至今對於不同 Nats 與及受質專一性的選擇的辨認機制仍然沒有明確的了解。所以想探討 NatA 與其他Nats 在受質的序列專一性，其辨認機制上的差別。SsArd1 源自於嗜熱性古細菌火山口硫化葉菌（Sulfolobus solfataricus），屬於 NatA 的蛋白質家族，催化特性偏好以絲胺酸 （serine）為起始的蛋白質受質，並且在 65oC 下具有最佳的酵素催化活性。利用蛋白質晶體學及繞射實驗得到解析度為 1.84 Å的 SsArd1 與受質胜肽複合體立體結構。與人類NatE 蛋白家族的 Naa50p 結構比較，發現此兩酵素座落於受質胜肽的第一個胺基酸附近的殘基（Glu35, SsArd1; Val29, Naa50p），其特性有著非常顯著的不同。將 Glu35突變後，酵素活性實驗證實了，SsArd1 受質專一性則替換成 NatE 的受質特性。 另外，分析不同空間群（space group）的SsArd1晶體結構，在β3與β4 之間的 loop 區域呈現多個構形的狀態，與常溫的Nat相比，此SsArd1上的loop長度也較為延伸，並且此 loop 上的兩個絲胺酸與在此區域的其他胺基酸形成複雜的氫鍵網路（hydrogen bond networks），所以想探討嗜熱性 Nat 具有長度延伸的loop 區域（β3與β4 之間）在熱穩定性上的功能與角色。熱穩定性實驗指出，兩個serine的單一定點突變和雙定點突變，與野生型（wild type）相比分別下降了3度與7度，此結果也與其酵素活性的降低有所關連。 再者，探討催化機制的部份，結構分析指出SsArd1上的 His88與Glu127 與其他已知結構 Nats 參與催化的胺基酸，在空間當中有非常相似的位置。另外，在結構當中，His88，Glu127 與受質之間存在一個穩定地水分子，此水分子參與受質第一個胺基酸上胺基的去質子化，並且協助乙醯化反應的完成。但此水分子如何在高溫環境下可以穩定存在與催化位中目前尚未清楚的了解。定點突變與酵素動力學實驗指出，單定點突變與雙定點突變，並不會影響期受質親和力 （Km），但卻讓酵素轉換能力（kcat）有不同程度的下降，甚至完全失去。然而，將His88，Glu127 胺基酸互換，卻可以讓 kcat 能力回復至與 wild type 相同。分析 Nats 參與催化胺基酸指出，這些催化的胺基酸並非為保守序列。結構分析指出 H88E/E127H 的兩胺基酸的側鏈與 wild type 相同，藉由 hydrogen bond 和水分子鍵結與穩定。綜合以上結果，蛋白質結晶學結合了光譜學與生化特性的研究，不僅詳細的解釋受質選擇專一性之辨認，並且也闡述古細菌中 SsArd1 熱穩定性與其酵素催化之機制。

Nα-acetyltransferases (Nats) possess a wide range of important biological functions. In recent studies, Nats also were applied to produce clinical drugs in large-scale including acetylation of thymosin α1 and thymosin β4 at N-terminus for maturation. The structure of Nats can vary according to the first two residues of their substrate. However, the mechanisms of substrate recognition of Nats are elusive. The Aim is identification of the mechanisms that NatA are able to preferentially acetylate sequence-specific substrates over other substrates from different Nats. SsArd1 from thermophilic Achaea Sulfolobus solfataricus, belonging to the NatA family with preference of Ser residues, exhibits the greatest activity of acetylation at optimal temperature of 65oC. Crystal structure of SsArd1 in complex with the peptide substrate was determined to 1.84 Å. Comparison of the structure of SsArd1 with human Naa50p (NatE) showed significant differences in key residues of enzymes near the first amino-acid position of the substrate peptide (Glu35 for SsArd1 and Val29 for Naa50p). The biochemical data revealed that the substrate specificity of SsArd1 could be altered the substrate of NatE by a range of Glu35 mutants. Additionally, the crystal structures of SsArd1 in different space groups indicated the loop region between β3 and β4 existing multiple conformations and extended loop compared with mesophilic Nats. Moreover, the loop of SsArd1 formed a hydrogen bond network via two Ser residues. We elucidate the functions of extended loop between β3 and β4 from thermophilic Nat. Comparing with wild-type SsArd1, the variants substituted with Ala (S75A, S82A and S75/S82A) and with loop deletion had almost identical folds. Strikingly, two single-point mutants showed ~3oC decrease in melting temperature, while two other variants showed even ~7oC decrease in melting temperature, which correlated to the seriously reducing enzymatic activity. Moreover, His88 and Glu127 of SsArd1 are located in very similar position of catalytic residues from structure-known Nats. In structural analysis, an ordered water was found between His88, Glu127 and substrate and performed deprotonation of the amino group from the first residue of protein substrate, facilitating the acetylation reaction. To understand why the ordered water molecule exists stably in the active site at high temperature, structure-based mutagenesis and kinetic studies were performed to indicated that substitution of His88 and Glu127 with Ala (H88A, E127A and H88/E127A) was loss of turnover rate although the binding ability has negligible effect on Km. However, the turnover rate could be rescued to wild-type level while the catalytic residues were exchanged each other. Sequence analysis indicates the catalytic residues from Nats are not conserved. The crystal structures of H88E/E127H mutated SsArd1 showed the side-chains of the two residues retain the hydrogen bonds with water. Taken together, the crystallographic studies combining spectroscopic and biochemical characterizations provide a detailed molecular basis for not only understanding the substrate-specific recognition, but also elucidating the mechanism of heat resistance and catalysis of the ancient archaeal SsArd1.