Expression, purification and characterization of recombinant phosphomannomutase and GDP-alpha-D-mannose pyrophosphorylase from Salmonella enterica, group B, for the synthesis of GDP-alpha-D-mannose from D-mannose.
The genes rfbK and rfbM from the rfb cluster (O-antigen biosynthesis) of Salmonella enterica, group B, encoding for the enzymes phosphomannomutase (EC 5.4.2.8) and GDP-alpha-D-mannose pyrophosphorylase (EC 2.7.7.13) were overexpressed in E.coli BL21 (DE3) with specific activities of 0.1 U/mg and 0.3-0.6 U/mg, respectively. Both enzymes were partially purified to give specific activities of 0.26 U/mg and 2.75 U/mg, respectively. Kinetic characterization of the homodimeric (108 kDa) GDP-alpha-D-mannose pyrophosphorylase revealed a K(m) for GTP and mannose-1-P of 0.2 mM and 0.01 mM with substrate surplus inhibition constants (Kis) of 10.9 mM and 0.7 mM, respectively. The product GDP-alpha-D-mannose gave a competitive inhibition with respect to GTP (Ki 14.7 microM) and an uncompetitive inhibition with respect to mannose-1-P (Ki 115 microM). Both recombinant enzymes were used for repetitive batch synthesis of GDP-alpha-D-mannose staring from D-mannose and GTP. In three subsequent batches 581 mg (960 mumol) GDP-alpha-D-mannose was synthesized with 80% average yield. The overall yield after product isolation was 22.9% (329 mumol, 199 mg).
Elling
L
L
Institut für Enzymtechnologie, Heinrich-Heine-Universität DAsseldorf im Forschungszentrum Jülich, Germany.
Ritter
J E
JE
Verseck
S
S
D004795:Enzyme Stability; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006155:Guanosine Diphosphate Mannose / Q000096:biosynthesis*; D007700:Kinetics; D009713:Nucleotidyltransferases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D017875:Phosphotransferases (Phosphomutases) / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012475:Salmonella / Q000201:enzymology*; D013379:Substrate Specificity
D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D006155:Guanosine Diphosphate Mannose; D009713:Nucleotidyltransferases; C026917:mannose 1-phosphate guanylyltransferase; D017875:Phosphotransferases (Phosphomutases); C052799:phosphomannomutase