Characterization of and functional evidence for Ste27 of Streptomyces sp. 139 as a novel spermine/spermidine acetyltransferase.
Ebosin, a novel exopolysaccharide produced by Streptomyces sp. 139, has remarkable anti-rheumatoid arthritis activity in vivo and its biosynthesis gene cluster (ste) consists of 27 ORFs (open reading frames). The present paper reports our study of the protein product encoded by ste27. Database searching reveals the homology of Ste27 with some spermidine/spermine acetyltransferases. To confirm the prediction, the ste27 gene was cloned and expressed in Escherichia coli BL21(DE3) cells and recombinant Ste27 was purified. The following enzymatic analysis revealed its ability of transferring the acetyl group from acetyl-CoA to spermidine and spermine, with spermidine being the preferred substrate. Ste27 can acetylate the N1, N4 and N8 positions on spermidine. The Km values of Ste27 were determined for spermidine and spermine, as well as for acetyl-CoA, poly-L-lysine and glucosamine 6-phosphate. Upon gene knockout, the exopolysaccharide-27m produced by the mutant strain Streptomyces sp. 139 (ste27-), compared with Ebosin, exhibited a significantly reduced binding activity to the interleukin-1 receptor. After gene complementation, the binding activity was partially restored. This demonstrated that the ste27 gene is involved in the biosynthesis of Ebosin. Molecular modelling was also carried out to predict the binding mode of Ste27 with acetyl-CoA, spermidine or spermine.
Key Laboratory of Biotechnology of Antibiotics, Ministry of Health, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
Yang
Y
Zhang
Jinming
J
Zhou
Ming
M
Chang
Liping
L
Bai
Junjie
J
Shan
Chen
C
Yao
Rong
R
Jiang
Lianhong
L
Guo
Ren
R
Zhang
Jiangbo
J
Wu
Yuan
Y
Li
D000123:Acetyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D055162:Biocatalysis; D003001:Cloning, Molecular; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature; D000099228:Diamine N-Acetyltransferase
D000123:Acetyltransferases; D001426:Bacterial Proteins; D017931:DNA Primers; D000099228:Diamine N-Acetyltransferase