title: Discovery and characterization of BlsE, a radical S-adenosyl-L-methionine decarboxylase involved in the blasticidin S biosynthetic pathway.
abstract: 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 Cys31, Cys35, Cys38 in the C×××C×MC motif and Gly73, Gly74, Glu75, Pro76 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.
authors.affiliation: State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Feng
authors.forename: Jun
authors.initials: J
authors.lastname: Wu
authors.forename: Nan
authors.initials: N
authors.lastname: Dai
authors.forename: Shuangjun
authors.initials: S
authors.lastname: Lin
authors.forename: H Howard
authors.initials: HH
authors.lastname: Xu
authors.forename: Zixin
authors.initials: Z
authors.lastname: Deng
authors.forename: Xinyi
authors.initials: X
authors.lastname: He
mesh_terms: D012437:Adenosylmethionine Decarboxylase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D053898:Biosynthetic Pathways / Q000235:genetics*; D019295:Computational Biology / Q000379:methods; D017931:DNA Primers / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D005418:Flavodoxin / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016296:Mutagenesis; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D009705:Nucleosides / Q000096:biosynthesis; D016415:Sequence Alignment; D013056:Spectrophotometry, Ultraviolet; D013302:Streptomyces / Q000201:enzymology*; D013997:Time Factors
chemical_list: D017931:DNA Primers; D005418:Flavodoxin; D009705:Nucleosides; C004500:blasticidin S; D009247:NADH, NADPH Oxidoreductases; C116041:flavodoxin NADPH oxidoreductase; D012437:Adenosylmethionine Decarboxylase