Identification of residues important for the activity of aldehyde-deformylating oxygenase through investigation into the structure-activity relationship

BMC Biotechnology

Research Article

Xuefeng Lu¹  Jian-Jun Li²  Qing Wang³  Luyao Bao³  Mei Li⁴  Chenjun Jia⁵ 

[1] Key Laboratory of Biofuels, Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, 266101, Qingdao, China;Key Laboratory of Biofuels, Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, 266101, Qingdao, China;Present Address: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, 100190, Beijing, China;Key Laboratory of Biofuels, Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, 266101, Qingdao, China;University of Chinese Academy of Sciences, 100049, Beijing, China;National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 100101, Beijing, China;University of Chinese Academy of Sciences, 100049, Beijing, China;National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 100101, Beijing, China;

关键词: Aldehyde-deformylating oxygenase;    Site-directed mutagenesis;    Structure-activity relationship;    Fatty alk(a/e)ne;    Synechococcus elongatus;    Synechocystis;   

DOI  :  10.1186/s12896-017-0351-8

 received in 2016-05-13, accepted in 2017-03-08,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundAldehyde-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 kcat value below 1 min−1, which would limit its application in biofuel production. To identify the activity related key residues of cADO is urgently required.ResultsThe 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 kcat 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%.ConclusionsSome important amino acids, which exerted some effects on cADO activity, were identified. Several enzyme variants exhibited greatly reduced activity, while the kcat 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.

【 授权许可】

CC BY   
© The Author(s). 2017