Cloning and characterization of long-chain fatty alcohol oxidase LjFAO1 in lotus japonicus.
The Lotus japonicus EST database was searched against Arabidopsis thaliana AtFAO3, a full-length cDNA that encodes a membrane-bound, flavin-containing, hydrogen peroxide generating, long-chain fatty alcohol oxidase. One EST fragment was detected, and the corresponding full-length cDNA was obtained by screening a cDNA library of L. japonicus. The LjFAO1 genomic DNA was amplified by PCR, to give a product 3.6 kb in length. Comparison between the LjFAO1 cDNA and genomic DNA revealed that the LjFAO1 contains 3 exons and 2 introns. RT-PCR analysis showed that the LjFAO1 was expressed in the whole plant, with the highest expression level in the apex and the lowest expression level in the siliques. The LjFAO1 gene was down-regulated by cold stress in both the apex and the cotelydon of the 8-day old seedlings, the first time that a long-chain alcohol oxidase has been shown to respond directly to stress. The full length cDNA and a C-terminal truncated version were overexpressed in Escherichia coli. The full length version of LjFAO1 exhibited long-chain fatty alcohol oxidase activity and was subsequently purified by Ni-NTA chromatography. The active LjFAO1 protein showed substrate specificities toward 1-dodecanol, 1-hexadecanol, and 1,16-hexadecanediol with Km values 59.6 +/- 14.8 (microM), 40.9 +/- 8.2 (microM) and 19.4 +/- 1.5 (microM), respectively, suggesting apparent differences in substrate preferences with AtFAO3.
Zhao
Shilan
S
School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai 200030, China.
Lin
Zhixin
Z
Ma
Wei
W
Luo
Da
D
Cheng
Qi
Q
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods*; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D000070116:Lotus / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*
D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase