Park Sun-Ha, Lee Chang Woo, Lee Sung Gu, Shin Seung Chul, Kim Hak Jun, Park Hyun, Lee Jun Hyuck
Unit of Polar Genomics, Korea Polar Research Institute, Incheon, Republic of Korea.
Department of Polar Sciences, University of Science and Technology, Incheon, Republic of Korea.
PLoS One. 2017 Jul 19;12(7):e0181705. doi: 10.1371/journal.pone.0181705. eCollection 2017.
Isoaspartyl dipeptidase (IadA) is an enzyme that catalyzes the hydrolysis of an isoaspartyl dipeptide-like moiety, which can be inappropriately formed in proteins, between the β-carboxyl group side chain of Asp and the amino group of the following amino acid. Here, we have determined the structures of an isoaspartyl dipeptidase (CpsIadA) from Colwellia psychrerythraea, both ligand-free and that complexed with β-isoaspartyl lysine, at 1.85-Å and 2.33-Å resolution, respectively. In both structures, CpsIadA formed an octamer with two Zn ions in the active site. A structural comparison with Escherichia coli isoaspartyl dipeptidase (EcoIadA) revealed a major difference in the structure of the active site. For metal ion coordination, CpsIadA has a Glu166 residue in the active site, whereas EcoIadA has a post-translationally carbamylated-lysine 162 residue. Site-directed mutagenesis studies confirmed that the Glu166 residue is critical for CpsIadA enzymatic activity. This residue substitution from lysine to glutamate induces the protrusion of the β12-α8 loop into the active site to compensate for the loss of length of the side chain. In addition, the α3-β9 loop of CpsIadA adopts a different conformation compared to EcoIadA, which induces a change in the structure of the substrate-binding pocket. Despite CpsIadA having a different active-site residue composition and substrate-binding pocket, there is only a slight difference in CpsIadA substrate specificity compared with EcoIadA. Comparative sequence analysis classified IadA-containing bacteria and archaea into two groups based on the active-site residue composition, with Type I IadAs having a glutamate residue and Type II IadAs having a carbamylated-lysine residue. CpsIadA has maximal activity at pH 8-8.5 and 45°C, and was completely inactivated at 60°C. Despite being isolated from a psychrophilic bacteria, CpsIadA is thermostable probably owing to its octameric structure. This is the first conclusive description of the structure and properties of a Type I IadA.
异天冬氨酰二肽酶（IadA）是一种催化异天冬氨酰二肽样部分水解的酶，该部分可在蛋白质中不适当形成，位于天冬氨酸的β-羧基侧链与下一个氨基酸的氨基之间。在此，我们分别以1.85 Å和2.33 Å的分辨率测定了来自嗜冷栖冷菌的异天冬氨酰二肽酶（CpsIadA）的结构，包括无配体结构以及与β-异天冬氨酰赖氨酸结合的复合物结构。在这两种结构中，CpsIadA均形成八聚体，活性位点含有两个锌离子。与大肠杆菌异天冬氨酰二肽酶（EcoIadA）的结构比较揭示了活性位点结构的主要差异。对于金属离子配位，CpsIadA在活性位点有一个谷氨酸166残基，而EcoIadA有一个翻译后氨甲酰化的赖氨酸162残基。定点诱变研究证实，谷氨酸166残基对CpsIadA的酶活性至关重要。从赖氨酸到谷氨酸的这种残基取代导致β12-α8环突出到活性位点，以补偿侧链长度的损失。此外，与EcoIadA相比，CpsIadA的α3-β9环采用不同的构象，这导致底物结合口袋的结构发生变化。尽管CpsIadA具有不同的活性位点残基组成和底物结合口袋，但与EcoIadA相比，CpsIadA的底物特异性仅有细微差异。比较序列分析根据活性位点残基组成将含有IadA的细菌和古菌分为两组，I型IadA具有谷氨酸残基，II型IadA具有氨甲酰化的赖氨酸残基。CpsIadA在pH 8 - 8.5和45°C时具有最大活性，在60°C时完全失活。尽管CpsIadA是从嗜冷细菌中分离出来的，但由于其八聚体结构，它具有热稳定性。这是对I型IadA结构和性质的首次确定性描述。
