Discovery and characterization of BlsE, a radical S-adenosyl-L-methionine decarboxylase involved in the blasticidin S biosynthetic pathway

PLoS One. 2013 Jul 18;8(7):e68545. doi: 10.1371/journal.pone.0068545. Print 2013.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosylmethionine Decarboxylase / chemistry*
  • Adenosylmethionine Decarboxylase / isolation & purification
  • Adenosylmethionine Decarboxylase / metabolism*
  • Amino Acid Sequence
  • Biosynthetic Pathways / genetics*
  • Computational Biology / methods
  • DNA Primers / genetics
  • Electron Spin Resonance Spectroscopy
  • Flavodoxin / metabolism
  • Kinetics
  • Molecular Sequence Data
  • Molecular Structure
  • Mutagenesis
  • NADH, NADPH Oxidoreductases / metabolism
  • Nucleosides / biosynthesis
  • Sequence Alignment
  • Spectrophotometry, Ultraviolet
  • Streptomyces / enzymology*
  • Time Factors

Substances

  • DNA Primers
  • Flavodoxin
  • Nucleosides
  • blasticidin S
  • NADH, NADPH Oxidoreductases
  • flavodoxin NADPH oxidoreductase
  • Adenosylmethionine Decarboxylase

Grant support

This study was supported in part by grants from the Ministry of Science and Technology of China (2012CB721004, 2009CB118901); the National Natural Science Foundation of China (31121064); Ministry of Education of China (20110073130011); Open Fund of State Key Laboratory of Bio-organic and Natural Products Chemistry; the Chen Xing Young Scholars Program, Shanghai Jiao Tong University; Dr Xu acknowledge Wang Family Faculty Fellowship from California State University, and U.S. Army Research Laboratory and the U.S. Army Research Office (W911NF-12-1-0059). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.