Wang Qing, Bao Luyao, Jia Chenjun, Li Mei, Li Jian-Jun, Lu Xuefeng
Key Laboratory of Biofuels, Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, 266101, China.
University of Chinese Academy of Sciences, Beijing, 100049, China.
BMC Biotechnol. 2017 Mar 16;17(1):31. doi: 10.1186/s12896-017-0351-8.
Aldehyde-deformylating oxygenase (ADO) is a key enzyme involved in the biosynthetic pathway of fatty alk(a/e)nes in cyanobacteria. However, cADO (cyanobacterial ADO) showed extreme low activity with the k value below 1 min, which would limit its application in biofuel production. To identify the activity related key residues of cADO is urgently required.
The amino acid residues which might affect cADO activity were identified based on the crystal structures and sequence alignment of cADOs, including the residues close to the di-iron center (Tyr39, Arg62, Gln110, Tyr122, Asp143 of cADO-1593), the protein surface (Trp 178 of cADO-1593), and those involved in two important hydrogen bonds (Gln49, Asn123 of cADO-1593, and Asp49, Asn123 of cADO-sll0208) and in the oligopeptide whose conformation changed in the absence of the di-iron center (Leu146, Asn149, Phe150 of cADO-1593, and Thr146, Leu148, Tyr150 of cADO-sll0208). The variants of cADO-1593 from Synechococcus elongatus PCC7942 and cADO-sll0208 from Synechocystis sp. PCC6803 were constructed, overexpressed, purified and kinetically characterized. The k values of L146T, Q49H/N123H/F150Y and W178R of cADO-1593 and L148R of cADO-sll0208 were increased by more than two-fold, whereas that of R62A dropped by 91.1%. N123H, Y39F and D143A of cADO-1593, and Y150F of cADO-sll0208 reduced activities by ≤ 20%.
Some important amino acids, which exerted some effects on cADO activity, were identified. Several enzyme variants exhibited greatly reduced activity, while the k values of several mutants are more than two-fold higher than the wild type. This study presents the report on the relationship between amino acid residues and enzyme activity of cADOs, and the information will provide a guide for enhancement of cADO activity through protein engineering.
醛脱甲酰基加氧酶（ADO）是蓝藻中脂肪链烯生物合成途径中的关键酶。然而，蓝藻ADO（cADO）活性极低，其k值低于1 min⁻¹，这将限制其在生物燃料生产中的应用。因此，迫切需要鉴定与cADO活性相关的关键残基。
基于cADOs的晶体结构和序列比对，鉴定了可能影响cADO活性的氨基酸残基，包括靠近双铁中心的残基（cADO-1593的Tyr39、Arg62、Gln110、Tyr122、Asp143）、蛋白质表面的残基（cADO-1593的Trp 178），以及参与两个重要氢键的残基（cADO-1593的Gln49、Asn123，cADO-sll0208的Asp49、Asn123）和在无双铁中心时构象发生变化的寡肽中的残基（cADO-1593的Leu146、Asn149、Phe150，cADO-sll0208的Thr146、Leu148、Tyr150）。构建了来自聚球藻属PCC7942的cADO-1593和来自集胞藻属PCC6803的cADO-sll0208的变体，进行了过表达、纯化及动力学表征。cADO-1593的L146T、Q49H/N123H/F150Y和W178R以及cADO-sll0208的L148R的k值增加了两倍以上，而R62A的k值下降了91.1%。cADO-1593的N123H、Y39F和D143A以及cADO-sll0208的Y150F的活性降低≤20%。
鉴定了一些对cADO活性有影响的重要氨基酸。几个酶变体的活性大幅降低，而几个突变体的k值比野生型高两倍以上。本研究报道了cADOs氨基酸残基与酶活性之间的关系，该信息将为通过蛋白质工程提高cADO活性提供指导。
