Discovery and characterization of BlsE, a radical S-adenosyl-L-methionine decarboxylase involved in the blasticidin S biosynthetic pathway.
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 char...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2013-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3715490?pdf=render |
| _version_ | 1819330818942697472 |
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| author | Jun Feng Jun Wu Nan Dai Shuangjun Lin H Howard Xu Zixin Deng Xinyi He |
| author_facet | Jun Feng Jun Wu Nan Dai Shuangjun Lin H Howard Xu Zixin Deng Xinyi He |
| author_sort | Jun Feng |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-24T14:04:35Z |
| format | Article |
| id | doaj.art-54c5d5507b4e4eac9ad4a1ead941e46f |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-24T14:04:35Z |
| publishDate | 2013-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-54c5d5507b4e4eac9ad4a1ead941e46f2022-12-21T16:52:23ZengPublic Library of Science (PLoS)PLoS ONE1932-62032013-01-0187e6854510.1371/journal.pone.0068545Discovery and characterization of BlsE, a radical S-adenosyl-L-methionine decarboxylase involved in the blasticidin S biosynthetic pathway.Jun FengJun WuNan DaiShuangjun LinH Howard XuZixin DengXinyi HeBlsE, 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.http://europepmc.org/articles/PMC3715490?pdf=render |
| spellingShingle | Jun Feng Jun Wu Nan Dai Shuangjun Lin H Howard Xu Zixin Deng Xinyi He Discovery and characterization of BlsE, a radical S-adenosyl-L-methionine decarboxylase involved in the blasticidin S biosynthetic pathway. PLoS ONE |
| title | Discovery and characterization of BlsE, a radical S-adenosyl-L-methionine decarboxylase involved in the blasticidin S biosynthetic pathway. |
| title_full | Discovery and characterization of BlsE, a radical S-adenosyl-L-methionine decarboxylase involved in the blasticidin S biosynthetic pathway. |
| title_fullStr | Discovery and characterization of BlsE, a radical S-adenosyl-L-methionine decarboxylase involved in the blasticidin S biosynthetic pathway. |
| title_full_unstemmed | Discovery and characterization of BlsE, a radical S-adenosyl-L-methionine decarboxylase involved in the blasticidin S biosynthetic pathway. |
| title_short | Discovery and characterization of BlsE, a radical S-adenosyl-L-methionine decarboxylase involved in the blasticidin S biosynthetic pathway. |
| title_sort | discovery and characterization of blse a radical s adenosyl l methionine decarboxylase involved in the blasticidin s biosynthetic pathway |
| url | http://europepmc.org/articles/PMC3715490?pdf=render |
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