Molecular Cloning and Sequencing of ADPglucose Pyrophosphorylase from Synechocystis PCC 6803

G. KAKEFUDA, Y-Y. CHARNG, A.A. IGLESIAS, L. MCINTOSH, J. PREISS

PLANT PHYSIOLOGY.

Año: 1992 vol. 99 p. 359 - 361

0032-0889

ADPGlc PPase3 is the regulatory enzyme for synthesis ofstarch in plants and glycogen in bacteria (8, 9). Previous workon cyanobacterial ADPGlc PPase has shown the enzyme tohave intermediate characteristics to that of the higher plantand bacterial enzymes (4). ADPGlc PPase from SynechocystisPCC 6803 is allosterically activated by 3-phosphoglycerateand inhibited by Pi, as are the higher plant enzymes. Thehomotetrameric structure of Synechocystis ADPGlc PPase issimilar to the enteric bacterial enzymes, which is in contrastwith the heterotetrameric nature of all higher plant enzymesstudied. Here we report the nucleotide sequence of ADPGlcPPase from Synechocystis PCC 6803. The Synechocystis clonewas isolated from a Synechocystis PCC 6803 genomic DNAlibrary. The probe used for screening the library was derivedfrom PCR amplification of genomic Synechocystis DNA.Amino acid sequences that were highly conserved in bothhigher plant and bacterial ADPGlc PPase sequences wereused to design degenerate primers for PCR amplification ofcyanobacterial DNA (Table I). Primer 1, which had a degeneracyof 512, was designed from the conserved amino acidsequences of the Escherichia coli ADPGlc PPase FBP activatorbinding site. The activator binding site determined for theE. coli enzyme is conserved in higher plants (7, 10). Theconservation occurs despite the fact that FBP does not activatehigher plant ADPGlc PPases. Primer 2, which had a degeneracyof 256, was designed from the conserved amino acidsequences (10) of the 8-azido-ADP-glucose affinity labelingsite previously determined in the E. coli enzyme (5). PCRamplification of genomic Synechocystis DNA with theseprimers generated a fragment of expected size. This fragmentwas used to isolate a clone from a genomic library.