Molecular Cloning and Sequencing of ADP-Glucose Pyrophosphorylase from Synechocystis PCC 6803

Abstract

ADPGlc PPase3 is the regulatory enzyme for synthesis of starch in plants and glycogen in bacteria (8, 9). Previous work on cyanobacterial ADPGlc PPase has shown the enzyme to have intermediate characteristics to that of the higher plant and bacterial enzymes (4). ADPGlc PPase from Synechocystis PCC 6803 is allosterically activated by 3-phosphoglycerate and inhibited by Pi, as are the higher plant enzymes. The homotetrameric structure of Synechocystis ADPGlc PPase is similar to the enteric bacterial enzymes, which is in contrast with the heterotetrameric nature of all higher plant enzymes studied. Here we report the nucleotide sequence of ADPGlc PPase from Synechocystis PCC 6803. The Synechocystis clone was isolated from a Synechocystis PCC 6803 genomic DNA library. The probe used for screening the library was derived from PCR amplification of genomic Synechocystis DNA. Amino acid sequences that were highly conserved in both higher plant and bacterial ADPGlc PPase sequences were used to design degenerate primers for PCR amplification of cyanobacterial DNA (Table I). Primer 1, which had a degeneracy of 512, was designed from the conserved amino acid sequences of the Escherichia coli ADPGlc PPase FBP activator binding site. The activator binding site determined for the E. coli enzyme is conserved in higher plants (7, 10). The conservation occurs despite the fact that FBP does not activate higher plant ADPGlc PPases. Primer 2, which had a degeneracy of 256, was designed from the conserved amino acid sequences (10) of the 8-azido-ADP-glucose affinity labeling site previously determined in the E. coli enzyme (5). PCR amplification of genomic Synechocystis DNA with these primers generated a fragment of expected size. This fragment was used to isolate a clone from a genomic library. The nucleotide and deduced amino acid sequence of Synechocystis ADPGlc PPase is shown in Figure 1. The first 39 N-terminal amino acids from the deduced sequence are in identity with the sequence determined by N-terminal sequencing of the purified protein from Synechocystis (our unpubTable I. Characteristics of ADP-Glucose Pyrophosphorylase Genomic DNA from Synechocystis PCC 6803