Cloning and characterization of GDP-perosamine synthetase (Per) from Escherichia coli O157:H7 and synthesis of GDP-perosamine in vitro.
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(m) and K(cat)/K(m) for GDP-4-keto-6-deoxy-d-mannose were 0.09 mM and 2.1 x 10(5)M(-1)S(-1), and those for l-glutamate were 2mM and 0.52 x 10(5)M(-1)S(-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.
Zhao
Guohui
G
State Key Laboratory of Microbial Technology, Shandong University, Jinan, Shandong 250100, China.
Liu
Jun
J
Liu
Xiang
X
Chen
Min
M
Zhang
Houcheng
H
Wang
Peng George
PG
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002238:Carbohydrate Epimerases / Q000235:genetics / Q000378:metabolism*; D002240:Carbohydrate Sequence; D003001:Cloning, Molecular; D019453:Escherichia coli O157 / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008358:Mannose / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011732:Pyridoxal Phosphate / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D000637:Transaminases / Q000235:genetics / Q000378:metabolism*
D000596:Amino Acids; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; C057033:4-amino-4,6-dideoxy-D-mannose; D011732:Pyridoxal Phosphate; D000637:Transaminases; D002238:Carbohydrate Epimerases; C061272:perosamine synthetase; D008358:Mannose