Kitagawa Masaki, Ito Nanako, Matsumoto Yuya, Saito Masaya, Tamura Takashi, Kusakabe Hitoshi, Inagaki Kenji, Imada Katsumi
Department of Macromolecular Science, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan.
Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University, Okayama 700-8530, Japan.
J Struct Biol X. 2021 Jan 13;5:100044. doi: 10.1016/j.yjsbx.2021.100044. eCollection 2021.
Harmuful proteins are usually synthesized as inactive precursors and are activated by proteolytic processing. l-Amino acid oxidase (LAAO) is a flavoenzyme that catalyzes the oxidative deamination of l-amino acid to produce a 2-oxo acid with ammonia and highly toxic hydrogen peroxide and, therefore, is expressed as a precursor. The LAAO precursor shows significant variation in size and the cleavage pattern for activation. However, the molecular mechanism of how the propeptide suppresses the enzyme activity remains unclear except for deaminating/decarboxylating l-phenylalanine oxidase (PAO), which has a short N-terminal propeptide composed of 14 residues. Here we show the inactivation mechanism of the l-lysine oxidase (LysOX) precursor (prLysOX), which has a long N-terminal propeptide composed of 77 residues, based on the crystal structure at 1.97 Å resolution. The propeptide of prLysOX indirectly changes the active site structure to inhibit the enzyme activity. prLysOX retains weak enzymatic activity with strict specificity for l-lysine and shows raised activity in acidic conditions. The structures of prLysOX crystals that soaked in a solution with various concentrations of l-lysine have revealed that prLysOX can adopt two conformations; one is the inhibitory form, and the other is very similar to mature LysOX. The propeptide region of the latter form is disordered, and l-lysine is bound to the latter form. These results indicate that prLysOX uses a different strategy from PAO to suppress the enzyme activity and suggest that prLysOX can be activated quickly in response to the environmental change without proteolytic processing.
有害蛋白质通常以无活性前体的形式合成，并通过蛋白水解加工被激活。L-氨基酸氧化酶（LAAO）是一种黄素酶，催化L-氨基酸的氧化脱氨反应，生成含氨的2-氧代酸和剧毒的过氧化氢，因此它以一种前体的形式表达。LAAO前体在大小和激活的切割模式上表现出显著差异。然而，除了对由14个残基组成的短N端前肽的L-苯丙氨酸氧化酶（PAO）进行脱氨/脱羧反应外，前肽抑制酶活性的分子机制仍不清楚。在此，我们基于分辨率为1.97 Å的晶体结构，展示了具有由77个残基组成的长N端前肽的L-赖氨酸氧化酶（LysOX）前体（prLysOX）的失活机制。prLysOX的前肽间接改变活性位点结构以抑制酶活性。prLysOX对L-赖氨酸具有严格特异性，保留较弱的酶活性，并在酸性条件下活性增强。浸泡在不同浓度L-赖氨酸溶液中的prLysOX晶体结构表明，prLysOX可以采取两种构象；一种是抑制形式，另一种与成熟的LysOX非常相似。后一种形式的前肽区域无序，且L-赖氨酸与后一种形式结合。这些结果表明，prLysOX采用了与PAO不同的策略来抑制酶活性，并表明prLysOX可以在不经过蛋白水解加工的情况下，快速响应环境变化而被激活。
