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 (K-iS) of 10.9 mM and 0.7 mM, respectively. The product GDP-alpha-D-mannose gave a competitive inhibition with respect to GTP (K-i 14.7 mu M) and an uncompetitive inhibition with respect to mannose-1-P (K-i 115 mu M). Both recombinant enzymes were used for repetitive batch synthesis of GDP-cw-D-mannose starting from D-mannose and GTP, In three subsequent batches 581 mg (960 mu mol) GDP-alpha-D-mannose was synthesized with 80% average yield, The overall yield after product isolation was 22.9% (329 mu mol, 199 mg).