Tracking Colanic Acid Repeat Unit Formation from Stepwise Biosynthesis Inactivation in Escherichia coli.
Tracking Colanic Acid Repeat Unit Formation from Stepwise Biosynthesis Inactivation in Escherichia coli.
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DOI：
10.1021/acs.biochem.1c00314
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发表时间：
2021-07-13
期刊：
影响因子：
2.9
通讯作者：
Troutman JM
中科院分区：
文献类型：
--
作者：
Reid AJ;Eade CR;Jones KJ;Jorgenson MA;Troutman JM
Colanic acid is a glycopolymer loosely associated with the outer membrane of Escherichia coli that plays a role in pathogen survival. For nearly six decades since its discovery, the functional identities of the enzymes necessary to synthesize colanic acid have yet to be assessed in full. Herein, we developed a method to detect the lipid-linked intermediates from each step of colanic acid biosynthesis in E. coli. The accumulation of each enzyme product was made possible by inactivating sequential genes involved in colanic acid biosynthesis and upregulating the colanic acid operon by inducing rcsA transcription. LC-MS analysis revealed that these accumulated materials were consistent with the well-documented composition analysis. Recapitulating the native bioassembly of colanic acid enabled us to identify the functional roles of the last two enzymes, WcaL and WcaK, associated with the formation of the lipid-linked oligosaccharide repeating unit of colanic acid. Importantly, biochemical evidence is provided for the formation of the final glycosylation hexasaccharide product formed by WcaL and the addition of a pyruvate moiety to form a pyruvylated hexasaccharide by WcaK. These findings provide insight toward the development of methods for the identification of enzyme functions during cell envelope synthesis.
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影响因子：
2.1
作者：
Kramer, NE;Smid, EJ;Breukink, E
通讯作者：
Breukink, E
影响因子：
3.2
作者：
GOTTESMAN, S;TRISLER, P;TORRESCABASSA, A
通讯作者：
TORRESCABASSA, A
影响因子：
2.8
作者：
Luo, Shen;McNeill, Megan;Levine, Rodney L.
通讯作者：
Levine, Rodney L.
影响因子：
3.2
作者：
Ranjit, Dev K.;Young, Kevin D.
通讯作者：
Young, Kevin D.
DOI：
10.1073/pnas.120163297
发表时间：
2000-06-06
影响因子：
11.1
作者：
Datsenko, KA;Wanner, BL
通讯作者：
Wanner, BL