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

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 A. 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.