title: Structure-oriented substrate specificity engineering of aldehyde-deformylating oxygenase towards aldehydes carbon chain length.
abstract: BACKGROUND
  Aldehyde-deformylating oxygenase (ADO) is an important enzyme involved in the biosynthetic pathway of fatty alk(a/e)nes in cyanobacteria. However, ADO exhibits quite low chain-length specificity with respect to the substrates ranging from C4 to C18 aldehydes, which is not suitable for producing fuels with different properties or different chain lengths.
  
  
  RESULTS
  Based on the crystal structures of cADOs (cyanobacterial ADO) with substrate analogs bound, some amino acids affecting the substrate specificity of cADO were identified, including the amino acids close to the aldehyde group and the hydrophobic tail of the substrate and those along the substrate channel. Using site-directed mutagenesis, selected amino acids were replaced with bulky ones introducing steric hindrance to the binding pocket via large functional groups. All mutants were overexpressed, purified and kinetically characterized. All mutants, except F87Y, displayed dramatically reduced activity towards C14,16,18 aldehydes. Notably, the substrate preferences of some mutants towards different chain-length substrates were enhanced: I24Y for n-heptanal, I27F for n-decanal and n-dodecanal, V28F for n-dodecanal, F87Y for n-decanal, C70F for n-hexanal, A118F for n-butanal, A121F for C4,6,7 aldehydes, V184F for n-dodecanal and n-decanal, M193Y for C6-10 aldehydes and L198F for C7-10 aldehydes. The impact of the engineered cADO mutants on the change of the hydrocarbon profile was demonstrated by co-expressing acyl-ACP thioesterase BTE, fadD and V184F in E. coli, showing that n-undecane was the main fatty alkane.
  
  
  CONCLUSIONS
  Some amino acids, which can control the chain-length selectivity of substrates of cADO, were identified. The substrate specificities of cADO were successfully changed through structure-guided protein engineering, and some mutants displayed different chain-length preference. The in vivo experiments of V184F in genetically engineered E. coli proved the importance of engineered cADOs on the distribution of the fatty alkane profile. The results would be helpful for the production of fatty alk(a/e)nes in cyanobacteria with different properties.
authors: Key Laboratory of Biofuels, Chinese Academy of Sciences, Qingdao, China ; University of Chinese Academy of Sciences, Beijing, 100049 China.
authors: Luyao
authors: L
authors: Bao
authors: Key Laboratory of Biofuels, Chinese Academy of Sciences, Qingdao, China ; National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, 1 North 2nd Street, Haidian District, Beijing, 100190 China.
authors: Jian-Jun
authors: JJ
authors: Li
authors: University of Chinese Academy of Sciences, Beijing, 100049 China ; National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing, 100101 China.
authors: Chenjun
authors: C
authors: Jia
authors: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing, 100101 China.
authors: Mei
authors: M
authors: Li
authors: Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao, 266101 China.
authors: Xuefeng
authors: X
authors: Lu
keywords: Aldehyde-deformylating oxygenase; Chain-length selectivity; Site-directed mutagenesis; Structure-guided protein engineering; Synechococcus elongatus PCC7942