Cloning and expression of the Escherichia coli glgC gene from a mutant containing an ADPglucose pyrophosphorylase with altered allosteric properties

Abstract

A mutant strain of Escherichia coli K-12, designated 618, accumulates glycogen at a faster rate than wild-type strain 356. The mutation affects the ADPglucose pyrophosphorylase regulatory properties (N. Creuzat-Sigal, M. Latil-Damotte, J. Cattaneo, and J. Puig, p. 647-680, in R. Piras and H. G. Pontis, ed., Biochemistry of the Glycocide Linkage, 1972). The enzyme is less dependent on the activator, fructose 1,6 bis-phosphate for activity and is less sensitive to inhibition by the inhibitor, 5'-AMP. The structural gene, glgC, for this allosteric mutant enzyme was cloned. into the bacterial plasmid pBR322 by inserting the chromosomal DNA at the PstI site. The glycogen biosynthetic genes were selected by cotransformation of the neighboring asd gene into an E. coli mutant also defective in branching enzyme (glgB) activity. Two recombinant plasmids, pEBLI and pEBL3, that had PstI chromosomal DNA inserts containing glgC and glgB were isolated. Branching enzyme and ADPglucose pyrophosphorylase activities were increased 240-and 40-fold, respectively, in the asdglgB mutant, E. coli K-12 6281. The E. coli K-12 618 mutant glgC gene product was characterized after transformation of an E. coli B ADPglucose pyrophosphorylase mutant with the recombinant plasmid pEBL3. The kinetic properties of the cloned ADPglucose pyrophosphorylase w.ere similar to those of the E. coli K-12 618 enzyme. The inserted DNA in pEBL1 was arranged in opposite orientation to that in pEBL3.

RapidRIP quantifies the intracellular metabolome of 7 industrial strains of E. coli

“…ADP-glucose (adpglc) is formed from glucose 1-phosphate via GLGC. Increased expression of glgC or mutations in glgC that increase GLGC activity have been shown to accumulate glycogen (Ballicora et al, 2003;Eydallin et al, 2007;Ghosh et al, 1992;Leung et al, 1986). No significant differences in the transcriptomic profiles of glgC in E. coli C were found and E. coli C is not known to harbor any unique mutation in the glgC gene, indicating that the elevated levels of adpglc could be due to kinetic factors.…”

Increasing maize seed weight by enhancing the cytoplasmic ADP-glucose pyrophosphorylase activity in transgenic maize plants

“…A mutant E. coli K12 strain, 618, accumulates 33% more glycogen than its wild-type as a result of an alteration in the regulatory properties of AGPase (Cattaneo et al 1969;Leung et al 1986;Lee et al 1987). This mutant glgC16 encodes a protein with a substitution of aspartic acid for glycine at position 336.…”

Mutagenesis of the potato ADPglucose pyrophosphorylase and characterization of an allosteric mutant defective in 3-phosphoglycerate activation.

“…By chemical modification approaches in combination with mutagenesis studies, residues involved in the binding of substrates ATP, G-1-P, and ADPglucose and of the allosteric effectors FBP and AMP have been identified in the Es-cherichia coli enzyme (7)(8)(9)(10)(11)(12)(13)(14)(15)(16). Pyridoxal 5-phosphate, which mimics the activator FBP, was observed to specifically react with Lys-39 in the E. coli enzyme, indicating that this residue lies in the allosteric domain of the bacterial enzyme (16).…”