Characterization of chimeric ADPglucose pyrophosphorylases of Escherichia coli and Agrobacterium tumefaciens. Importance of the C-terminus on the selectivity for allosteric regulators.
ADPglucose pyrophosphorylase catalyzes the regulatory step in the pathway for bacterial glycogen synthesis. The enzymes from different organisms exhibit distinctive regulatory properties related to the main carbon metabolic pathway. Escherichia coli ADPglucose pyrophosphorylase is mainly activated by fructose 1,6-bisphosphate (FBP), whereas the Agrobacterium tumefaciens enzyme is activated by fructose 6-phosphate (F6P) and pyruvate. Little is known about the regions determining the specificity for the allosteric regulator. To study the function of different domains, two chimeric enzymes were constructed. "AE" contains the N-terminus (271 amino acids) of the A. tumefaciens ADPglucose pyrophosphorylase and the C-terminus (153 residues) of the E. coli enzyme, and "EA", the inverse construction. Expression of the recombinant wild-type and chimeric enzymes was performed using derivatives of the pET24a plasmid. Characterization of the purified chimeric enzymes showed that the C-terminus of the E. coli enzyme is relevant for the selectivity by FBP. However, this region seems to be less important for the specificity by F6P in the A. tumefaciens enzyme. The chimeric enzyme AE is activated by both FBP and F6P, neither of which affect EA. Pyruvate activates EA with higher apparent affinity than AE, suggesting that the C-terminus of the A. tumefaciens enzyme plays a role in the binding of this effector. The allosteric inhibitor site is apparently disrupted, as a marked desensitization toward AMP was observed in the chimeric enzymes.
Ballicora
Miguel A
MA
Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824, USA.
Sesma
Juliana I
JI
Iglesias
Alberto A
AA
Preiss
Jack
J
D016960:Agrobacterium tumefaciens / Q000201:enzymology*; D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology*; D051599:Glucose-1-Phosphate Adenylyltransferase; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D009713:Nucleotidyltransferases; D051599:Glucose-1-Phosphate Adenylyltransferase