Cloning, expression, and sequence of an allosteric mutant ADPglucose pyrophosphorylase from Escherichia coli B.
Escherichia coli B mutant strain SG14 accumulates glycogen at 28% of the rate observed for the parent strain. This is due to the presence of an ADPglucose pyrophosphorylase with altered allosteric properties including lower apparent affinities for substrates, the activator, fructose-1,6-bisphosphate, and the inhibitor, AMP. The mutant enzyme also is completely insensitive to activation by NADPH. To clone this mutant, an SG14 library was constructed by insertion of the chromosomal DNA into the PstI site of pBR322. Screening of the library via colony hybridization with a wild-type gene (glgC) probe resulted in the successful isolation of a recombinant plasmid, designated pPJ2, which contained the mutant glgC gene. The enzyme expressed from pPJ2 was partially purified and found to be very similar in kinetic and allosteric properties to the enzyme isolated from the SG14 strain. The mutant glgC gene, a HincII fragment from pPJ2, was then subcloned into pUC118/119 for dideoxy sequencing of both strands. One amino acid change was found in a region that is highly conserved in all known sequences: a single point mutation at the deduced amino acid residue 44 resulted in a change of alanine to threonine. The properties of this mutant are discussed in comparison to other known allosteric mutants.
Department of Biochemistry, Michigan State University, East Lansing 48824.
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Meyer
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Ghosh
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Nadler
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Preiss
D000249:Adenosine Monophosphate / Q000494:pharmacology; D000494:Allosteric Regulation / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005635:Fructosediphosphates / Q000494:pharmacology; D015870:Gene Expression; D051599:Glucose-1-Phosphate Adenylyltransferase; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D009249:NADP / Q000494:pharmacology; D009693:Nucleic Acid Hybridization; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics*; D010728:Phosphoenolpyruvate / Q000494:pharmacology; D010957:Plasmids; D014169:Transformation, Bacterial
D005635:Fructosediphosphates; D000249:Adenosine Monophosphate; D009249:NADP; D010728:Phosphoenolpyruvate; D009713:Nucleotidyltransferases; D051599:Glucose-1-Phosphate Adenylyltransferase; D008274:Magnesium; C029063:fructose-1,6-diphosphate