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Discovery and Characterization of BlsE,a Radical S-Adenosyl-L-methionine Decarboxylase Involved in the Blasticidin S Biosynthetic Pathway
Authors:Jun Feng  Jun Wu  Nan Dai  Shuangjun Lin  H Howard Xu  Zixin Deng  Xinyi He
Institution:1. State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China.; 2. New England Biolabs, Inc., Research Department, Ipswich, Massachusetts, United States of America.; 3. Department of Biological Sciences, California State University Los Angeles, Los Angeles, California, United States of America.; University of Nottingham, United Kingdom,
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.
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