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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 C×××C×MC motif and Gly 73 , Gly 74 , Glu 75 , Pro 76  in the GGEP motif were crucial amino acids for BlsE activity while mutation of Met 37  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.

Discovery and Characterization of BlsE, a Radical S-Adenosyl-L-methionine Decarboxylase Involved in the Blasticidin S Biosynthetic Pathway