Journal
PLOS ONE
Volume 8, Issue 7, Pages -Publisher
PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.pone.0068545
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Funding
- Ministry of Science and Technology of China [2012CB721004, 2009CB118901]
- National Natural Science Foundation of China [31121064]
- Ministry of Education of China [20110073130011]
- State Key Laboratory of Bio-organic and Natural Products Chemistry
- Shanghai Jiao Tong University
- California State University
- U.S. Army Research Laboratory
- U.S. Army Research Office [W911NF-12-1-0059]
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BlsE, a predicted radical S-adenosyl-L-methionine (SAM) protein, was anaerobically purified and reconstituted in vitro to study its function in the blasticidin S biosynthetic pathway. The putative role of BlsE was elucidated based on bioinformatics analysis, genetic inactivation and biochemical characterization. Biochemical results showed that BlsE is a SAM-dependent radical enzyme that utilizes cytosylglucuronic acid, the accumulated intermediate metabolite in blsE mutant, as substrate and catalyzes decarboxylation at the C5 position of the glucoside residue to yield cytosylarabinopyranose. Additionally, we report the purification and reconstitution of BlsE, characterization of its [4Fe-4S] cluster using UV-vis and electron paramagnetic resonance (EPR) spectroscopic analysis, and investigation of the ability of flavodoxin (Fld), flavodoxin reductase (Fpr) and NADPH to reduce the [4Fe-4S](2+) cluster. Mutagenesis studies demonstrated that Cys(31), Cys(35), Cys(38) in the CxxxCxMC motif and Gly(73), Gly(74), Glu(75), Pro(76) in the GGEP motif were crucial amino acids for BlsE activity while mutation of Met37 had little effect on its function. Our results indicate that BlsE represents a typical [4Fe-4S]-containing radical SAM enzyme and it catalyzes decarboxylation in blasticidin S biosynthesis.
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