Kinetic properties of the glucose-6-phosphate and 6-phosphogluconate dehydrogenases from Corynebacterium glutamicum and their application for predicting pentose phosphate pathway flux in vivo.
The glucose-6-phosphate (Glc6P) and 6-phosphogluconate (6PG) dehydrogenases of the amino-acid-producing bacterium Corynebacterium glutamicum were purified to homogeneity and kinetically characterized. The Glc6P dehydrogenase was a heteromultimeric complex, which consists of Zwf and OpcA subunits. The product inhibition pattern of the Glc6P dehydrogenase was consistent with an ordered bi-bi mechanism. The 6PG dehydrogenase was found to operate according to a Theorell-Chance ordered bi-ter mechanism. Both enzymes were inhibited by NADPH and the 6PG dehydrogenase additionally by ATP, fructose 1,6-bisphosphate (Fru1,6P2), D-glyceraldehyde 3-phosphate (Gra3P), erythrose 4-phosphate and ribulose 5-phosphate (Rib5P). The inhibition by NADPH was considered to be most important, with inhibition constants of around 25 microM for both enzymes. Intracellular metabolite concentrations were determined in two isogenic strains of C. glutamicum with plasmid-encoded NAD- and NADP-dependent glutamate dehydrogenases. NADP+ and NADPH levels were between 130 microM and 290 microM, which is very much higher than the respective Km and Ki values. The Glc6P concentration was around 500 microM in both strains. The in vivo fluxes through the oxidative part of the pentose phosphate pathway calculated on the basis of intracellular metabolite concentrations and the kinetic constants of the purified enzymes determined in vitro were in agreement with the same fluxes determined by NMR after 13C-labelling. From the derived kinetic model thus validated, it is concluded that the oxidative pentose phosphate pathway in C. glutamicum is mainly regulated by the ratio of NADPH and NADP+ concentrations and the specific enzyme activities of both dehydrogenases.
Moritz
B
B
Institut für Biotechnologie I, Jülich, Germany.
Striegel
K
K
De Graaf
A A
AA
Sahm
H
H
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D002247:Carbon Isotopes; D003352:Corynebacterium / Q000378:metabolism*; D005635:Fructosediphosphates / Q000378:metabolism; D005954:Glucosephosphate Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism*; D005969:Glutamate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D005986:Glyceraldehyde 3-Phosphate / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009154:Mutation; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010427:Pentose Phosphate Pathway; D010734:Phosphogluconate Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism*; D012274:Ribulosephosphates / Q000378:metabolism; D013403:Sugar Phosphates / Q000378:metabolism
D002247:Carbon Isotopes; D005635:Fructosediphosphates; D012274:Ribulosephosphates; D013403:Sugar Phosphates; D009243:NAD; D005986:Glyceraldehyde 3-Phosphate; C031524:ribulose 5-phosphate; D009249:NADP; C026959:erythrose 4-phosphate; D000255:Adenosine Triphosphate; D010734:Phosphogluconate Dehydrogenase; D005954:Glucosephosphate Dehydrogenase; D005969:Glutamate Dehydrogenase