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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. 1996

L Elling, and J E Ritter, and S Verseck
Institut für Enzymtechnologie, Heinrich-Heine-Universität DAsseldorf im Forschungszentrum Jülich, Germany.

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).

UI MeSH Term Description Entries
D007700 Kinetics The rate dynamics in chemical or physical systems.
D009713 Nucleotidyltransferases A class of enzymes that transfers nucleotidyl residues. EC 2.7.7. Nucleotidyltransferase
D011994 Recombinant Proteins Proteins prepared by recombinant DNA technology. Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D004795 Enzyme Stability The extent to which an enzyme retains its structural conformation or its activity when subjected to storage, isolation, and purification or various other physical or chemical manipulations, including proteolytic enzymes and heat. Enzyme Stabilities,Stabilities, Enzyme,Stability, Enzyme
D006153 Guanosine Diphosphate A guanine nucleotide containing two phosphate groups esterified to the sugar moiety. GDP,Guanosine 5'-Diphosphate,Guanosine 5'-Trihydrogen Diphosphate,5'-Diphosphate, Guanosine,5'-Trihydrogen Diphosphate, Guanosine,Diphosphate, Guanosine,Diphosphate, Guanosine 5'-Trihydrogen,Guanosine 5' Diphosphate,Guanosine 5' Trihydrogen Diphosphate
D006155 Guanosine Diphosphate Mannose A nucleoside diphosphate sugar which can be converted to the deoxy sugar GDPfucose, which provides fucose for lipopolysaccharides of bacterial cell walls. Also acts as mannose donor for glycolipid synthesis. GDP Mannose,Guanosine Diphosphomannose,Guanosine Pyrophosphate Mannose,Diphosphate Mannose, Guanosine,Diphosphomannose, Guanosine,Mannose, GDP,Mannose, Guanosine Diphosphate,Mannose, Guanosine Pyrophosphate,Pyrophosphate Mannose, Guanosine
D012475 Salmonella A genus of gram-negative, facultatively anaerobic, rod-shaped bacteria that utilizes citrate as a sole carbon source. It is pathogenic for humans, causing enteric fevers, gastroenteritis, and bacteremia. Food poisoning is the most common clinical manifestation. Organisms within this genus are separated on the basis of antigenic characteristics, sugar fermentation patterns, and bacteriophage susceptibility.
D013379 Substrate Specificity A characteristic feature of enzyme activity in relation to the kind of substrate on which the enzyme or catalytic molecule reacts. Specificities, Substrate,Specificity, Substrate,Substrate Specificities
D017875 Phosphotransferases (Phosphomutases) A group of enzymes that catalyze an intramolecular transfer of a phosphate group. It has been shown in some cases that the enzyme has a functional phosphate group, which can act as the donor. These were previously listed under PHOSPHOTRANSFERASES (EC 2.7.-). (From Enzyme Nomenclature, 1992) EC 5.4.2. Phosphomutases

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