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.

Abstract

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).