Partial purification and characterization of a non-cyanobacterial cyanophycin synthetase from Acinetobacter calcoaceticus strain ADP1 with regard to substrate specificity, substrate affinity and binding to cyanophycin.
This study reports, for the first time, purification and biochemical characterization of a cyanophycin synthetase from a non-cyanobacterial strain. Cyanophycin synthetase of Acinetobacter calcoaceticus strain ADP1 was purified 69-fold from recombinant Escherichia coli by two chromatographic steps and one novel affinity step utilizing the Mg(2+)-dependent binding of the enzyme to cyanophycin. Unlike cyanobacterial cyanophycin synthetases characterized so far, the purified enzyme from A. calcoaceticus strain ADP1 did not accept lysine as an alternative substrate to arginine. The apparent K(m)-values for arginine (47 microM) and aspartic acid (240 microM) were similar to those of known cyanophycin synthetases from cyanobacteria, but this enzyme had a slightly higher affinity for aspartic acid. In addition, the two different ATP-binding sites of the enzyme were characterized independently of each other with respect to K(m) values for ATP. The ATP-binding site responsible for the addition of arginine was found to have a much higher affinity for ATP (38 microM) than that responsible for the addition of aspartate (210 mM). Furthermore, the binding of the enzyme to the two possible forms of cyanophycin granule polypeptide (CGP), CGP-Asp and CGP-Arg, was studied. While both forms bound around 30-40 % of the enzyme activity present under the assay conditions, binding was Mg(2+)-dependent in the case of CGP-Asp. Two-dimensional gel electrophoresis revealed that both forms of cyanophycin were equally abundant in cyanophycin-accumulating cells of A. calcoaceticus ADP1.
Krehenbrink
Martin
M
Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Corrensstraße 3, D-48149 Münster, Germany.
Steinbüchel
Alexander
A
Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Corrensstraße 3, D-48149 Münster, Germany.
D016954:Acinetobacter calcoaceticus / Q000201:enzymology* / Q000235:genetics; D000255:Adenosine Triphosphate / Q000378:metabolism; D001120:Arginine / Q000378:metabolism; D001224:Aspartic Acid / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D007700:Kinetics; D010453:Peptide Synthases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D010940:Plant Proteins; D011994:Recombinant Proteins; C024250:cyanophycin; D001224:Aspartic Acid; D000255:Adenosine Triphosphate; D001120:Arginine; D010453:Peptide Synthases; C113257:cyanophycin synthase, bacteria