BIOSYNTHESIS OF THE ESCHERICHIA-COLI SIDEROPHORE ENTEROBACTIN - SEQUENCE OF THE ENTF GENE, EXPRESSION AND PURIFICATION OF ENTF, AND ANALYSIS OF COVALENT PHOSPHOPANTETHEINE

被引:138
作者
RUSNAK, F [1 ]
SAKAITANI, M [1 ]
DRUECKHAMMER, D [1 ]
REICHERT, J [1 ]
WALSH, CT [1 ]
机构
[1] HARVARD UNIV,SCH MED,DEPT BIOL CHEM & MOLEC PHARMACOL,BOSTON,MA 02115
关键词
D O I
10.1021/bi00225a027
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The sequence of the entF gene which codes for the serine activating enzyme in enterobactin biosynthesis is reported. The gene encodes a protein with a calculated molecular weight of 142 006 and shares homologies with the small subunits of gramicidin S synthetase and tyrocidine synthetase. We have subcloned and overexpressed entF in a multicopy plasmid and attempted to demonstrate L-serine-dependent ATP-[P-32]PP(i) exchange activity and its participation in enterobactin biosynthesis, but the overexpressed enzyme appears to be essentially inactive in crude extract. A partial purification of active EntF from wild-type Escherichia coli, however, has confirmed the expected activities of EntF. In a search for possible causes for the low level of activity of the overexpressed enzyme, we have discovered that EntF contains a covalently bound phosphopantetheine cofactor.
引用
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页码:2916 / 2927
页数:12
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