Pan Guohui, Gao Xiaoqin, Fan Keqiang, Liu Junlin, Meng Bing, Gao Jinmin, Wang Bin, Zhang Chaobo, Han Hui, Ai Guomin, Chen Yihua, Wu Dong, Liu Zhi-Jie, Yang Keqian
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences , Beijing 100101, People's Republic of China.
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences , Beijing 100101, People's Republic of China.
ACS Chem Biol. 2017 Jan 20;12(1):142-152. doi: 10.1021/acschembio.6b00621. Epub 2016 Nov 30.
C-C bond ring cleaving oxygenases represent a unique family of enzymes involved in the B ring cleavage reaction only observed in atypical angucycline biosynthesis. B ring cleavage is the key reaction leading to dramatic divergence in the final structures of atypical angucyclines. Here, we present the crystal structure of AlpJ, the first structure of this family of enzymes. AlpJ has been verified as the enzyme catalyzing C-C bond cleavage in kinamycin biosynthesis. The crystal structure of the AlpJ monomer resembles the dimeric structure of ferredoxin-like proteins. The N- and C-terminal halves of AlpJ are homologous, and both contain a putative hydrophobic substrate binding pocket in the "closed" and "open" conformations, respectively. Structural comparison of AlpJ with ActVA-Orf6 and protein-ligand docking analysis suggest that the residues including Asn60, Trp64, and Trp181 are possibly involved in substrate recognition. Site-directed mutagenesis results supported our hypothesis, as mutation of these residues led to nearly a complete loss of the activity of AlpJ. Structural analysis also revealed that AlpJ possesses an intramolecular domain-domain interface, where the residues His50 and Tyr178 form a hydrogen bond that probably stabilizes the three-dimensional structure of AlpJ. Site-directed mutagenesis showed that the two residues, His50 and Tyr178, were vital for the activity of AlpJ. Our findings shed light on the structure and catalytic mechanism of the AlpJ family of oxygenases, which presumably involves two active sites that might function in a cooperative manner.
C-C键环裂解加氧酶代表了一类独特的酶家族，它们参与仅在非典型安古环素生物合成中观察到的B环裂解反应。B环裂解是导致非典型安古环素最终结构产生显著差异的关键反应。在此，我们展示了AlpJ的晶体结构，这是该酶家族的首个结构。AlpJ已被证实是在金霉素生物合成中催化C-C键裂解的酶。AlpJ单体的晶体结构类似于铁氧还蛋白样蛋白的二聚体结构。AlpJ的N端和C端部分是同源的，并且在“闭合”和“开放”构象中分别包含一个假定的疏水底物结合口袋。AlpJ与ActVA-Orf6的结构比较以及蛋白质-配体对接分析表明，包括Asn60、Trp64和Trp181在内的残基可能参与底物识别。定点诱变结果支持了我们的假设，因为这些残基的突变导致AlpJ的活性几乎完全丧失。结构分析还表明，AlpJ具有分子内结构域-结构域界面，其中His50和Tyr178残基形成氢键，这可能稳定了AlpJ的三维结构。定点诱变表明，His50和Tyr178这两个残基对AlpJ的活性至关重要。我们的研究结果揭示了AlpJ加氧酶家族的结构和催化机制，推测其涉及两个可能协同发挥作用的活性位点。
