Cloning and characterization of GDP-perosamine synthetase (Per) from Escherichia coli O157:H7 and synthesis of GDP-perosamine in vitro
- State Key Laboratory of Microbial Technology, Shandong University, Jinan, Shandong 250100 (China)
GDP-perosamine synthetase (Per, E.C. not yet classified) is important to the synthesis of Escherichia coli O157:H7 O-antigen. The mutant in per gene can disrupt the synthesis of O157 O-antigen. In this study, GDP-perosamine synthetase was cloned from E. coli O157:H7 and over-expressed in E. coli BL21 (DE3). The recombinant His-tagged Per fusion protein was a decamer with molecular weight of 431 kDa. The optimal pH value of this recombinant protein was 7.5. The divalent ions had no significant effect on Per-catalyzed reaction. The K{sub m} and K{sub cat}/K{sub m} for GDP-4-keto-6-deoxy-D-mannose were 0.09 mM and 2.1 x 10{sup 5} M{sup -1} S{sup -1}, and those for L-glutamate were 2 mM and 0.52 x 10{sup 5} M{sup -1}S{sup -1}, respectively. Per was used to synthesize GDP-perosamine from GDP-mannose together with recombinant GDP-mannose dehydratase (GMD, E.C. 4.2.1.47). The purified GDP-perosamine was identified by MS and NMR. In summary, this work provided a feasible approach for the synthesis of GDP-perosamine which can lead to the study of LPS biosynthesis of pathogenic E. coli O157:H7.
- OSTI ID:
- 21032979
- Journal Information:
- Biochemical and Biophysical Research Communications, Journal Name: Biochemical and Biophysical Research Communications Journal Issue: 3 Vol. 363; ISSN 0006-291X; ISSN BBRCA9
- Country of Publication:
- United States
- Language:
- English
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