Fructose-1,6-bisphosphatase from Corynebacterium glutamicum: expression and deletion of the fbp gene and biochemical characterization of the enzyme.
The class II fructose-1,6-bisphosphatase gene of Corynebacterium glutamicum, fbp, was cloned and expressed with a N-terminal His-tag in Escherichia coli. Purified, His-tagged fructose-1,6-bisphosphatase from C. glutamicum was shown to be tetrameric, with a molecular mass of about 140 kDa for the homotetramer. The enzyme displayed Michaelis-Menten kinetics for the substrate fructose 1,6-bisphosphate with a K(m) value of about 14 micro M and a V(max) of about 5.4 micro mol min(-1) mg(-1) and k(cat )of about 3.2 s(-1). Fructose-1,6-bisphosphatase activity was dependent on the divalent cations Mg(2+) or Mn(2+) and was inhibited by the monovalent cation Li(+) with an inhibition constant of 140 micro M. Fructose 6-phosphate, glycerol 3-phosphate, ribulose 1,5-bisphosphate and myo-inositol-monophosphate were not significant substrates of fructose-1,6-bisphosphatase from C. glutamicum. The enzymatic activity was inhibited by AMP and phosphoenolpyruvate and to a lesser extent by phosphate, fructose 6-phosphate, fructose 2,6-bisphosphate, and UDP. Fructose-1,6-bisphosphatase activities and protein levels varied little with respect to the carbon source. Deletion of the chromosomal fbp gene led to the absence of any detectable fructose-1,6-bisphosphatase activity in crude extracts of C. glutamicum WTDelta fbp and to an inability of this strain to grow on the carbon sources acetate, citrate, glutamate, and lactate. Thus, fbp is essential for growth on gluconeogenic carbon sources and likely codes for the only fructose-1,6-bisphosphatase in C. glutamicum.
Rittmann
Doris
D
Institute of Biotechnology 1, Research Center Jülich, 52425, Jülich, Germany.
Schaffer
Steffen
S
Wendisch
Volker F
VF
Sahm
Hermann
H
D003352:Corynebacterium / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D006597:Fructose-Bisphosphatase / Q000235:genetics* / Q000378:metabolism; D017353:Gene Deletion; D015870:Gene Expression; D013379:Substrate Specificity
D006597:Fructose-Bisphosphatase