Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo,Tokyo 113-0033, Japan.
Bioorg Med Chem Lett. 2012 Sep 15;22(18):5784-90. doi: 10.1016/j.bmcl.2012.07.102. Epub 2012 Aug 7.
A cDNA encoding a novel copper amine oxidase (CAO) was cloned and sequenced from the Chinese club moss Huperzia serrata (Huperziaceae), which produces the Lycopodium alkaloid huperzine A. A 2043-bp open reading frame encoded an Mr 76,854 protein with 681 amino acids. The deduced amino acid sequence shared 44-56% identity with the known CAOs of plant origin, and contained the active site consensus sequence of Asn-Tyr-Asp/Glu. The phylogenetic tree analysis revealed that HsCAO from the primitive vascular plant H. serrata is closely related to Physcomitrella patens subsp CAO. The recombinant enzyme, heterologously expressed in Escherichia coli, catalyzed the oxidative deamination of aliphatic and aromatic amines. Among them, the enzyme accepted cadaverine as the best substrate to catalyze the oxidative deamination to Δ(1)-piperideine, which is the precursor of the Lycopodium alkaloids. Furthermore, a homology modeling and site-directed mutagenesis studies predicted the active site architecture, which suggested the crucial active site residues for the observed substrate preference. This is the first report of the cloning and characterization of a CAO enzyme from the primitive Lycopodium plant.
从中国石松（石松科）中克隆并测序了编码一种新型铜胺氧化酶（CAO）的 cDNA，该植物产生石松生物碱石杉碱 A。2043 个碱基对的开放阅读框编码了一个 Mr 76854 的蛋白质，包含 681 个氨基酸。推导的氨基酸序列与已知的植物来源的 CAO 具有 44-56%的同一性，并且包含活性位点保守序列 Asn-Tyr-Asp/Glu。系统发育树分析表明，来自原始维管植物石松的 HsCAO 与拟南芥 subsp CAO 密切相关。在大肠杆菌中异源表达的重组酶催化脂肪族和芳香族胺的氧化脱氨。其中，该酶以腐胺为最佳底物接受，催化氧化脱氨生成Δ(1)-哌啶，这是石松生物碱的前体。此外，同源建模和定点突变研究预测了活性位点结构，这表明了观察到的底物偏好的关键活性位点残基。这是首次从原始石松植物中克隆和表征 CAO 酶。
