Expression, purification and characterization of recombinant phosphomannomutase and GDP-α-D-mannose pyrophosphorylase from Salmonella enterica, group B, for the synthesis of GDP-α-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 GDPa-D-mannose pyrophosphorylase (EC 2.7.7.13) were overexpressed in E.coli BL21 (DE3) with specific activities of 0.1 U/mg and 03-0.6 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-a-D-mannose pyrophosphorylase revealed a Km for GTP and mannose-1-P of 0.2 mM and 0.01 mM with substrate surplus inhibition constants (K^ of 10.9 mM and 0.7 mM, respectively. The product GDP-a-D-mannose gave a competitive inhibition with respect to GTP (K| 14.7 uM) and an uncompetitive inhibition with respect to mannose-1-P (K, 115 uM). Both recombinant enzymes were used for repetitive batch synthesis of GDP-a-D-mannose starting from D-mannose and GTP. In three subsequent batches 581 mg (960 umol) GDP-a-Dmannose was synthesized with 80% average yield. The overall yield after product isolation was 22.9% (329 umol, 199 mg).