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.
Highlights
Blasticidin S (1), a representative peptidyl nucleoside antibiotic produced by Streptomyces griseochromogenes, exhibits strong inhibitory activity against rice blast caused by Pyricularia oryzae Cavara in most regions of Asia and has replaced mercury fungicides [1]
The content of iron and sulfur indicated that BlsE may contain two [4Fe–4S] clusters, which is supported by the fact that removal of two electrons is theoretically necessary for the oxidative decarboxylation of CGA to obtain CAP
We assume that the C31666C356M37C38 motif binds one of the [4Fe–4S] clusters which coordinates binding with SAM, while another motif (C3176C31966C322) may provide the binding site for the second iron-sulfur that is supposed to coordinate with the nucleophilic groups of CGA
Summary
Blasticidin S (1), a representative peptidyl nucleoside antibiotic produced by Streptomyces griseochromogenes, exhibits strong inhibitory activity against rice blast caused by Pyricularia oryzae Cavara in most regions of Asia and has replaced mercury fungicides [1] It inhibits protein synthesis in both prokaryotes and eukaryotes through inhibition of peptide-bond formation in the ribosomal machinery [2]. Compound 1 and its resistance gene (bsr) have been widely used for transgenic selection in eukaryotic cells [3,4,5,6] Unlike typical nucleosides, such as puromycin, nikkomycin, and pacidamycin, 1 features a pyranoside core moiety that is shared by arginomycin (2), mildiomycin (3), and cytomycin (4) (Figure 1A). The three protein homologs were predicted to be radical S-adenosyl-L-
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