1887

Abstract

This study reports, for the first time, purification and biochemical characterization of a cyanophycin synthetase from a non-cyanobacterial strain. Cyanophycin synthetase of strain ADP1 was purified 69-fold from recombinant by two chromatographic steps and one novel affinity step utilizing the Mg-dependent binding of the enzyme to cyanophycin. Unlike cyanobacterial cyanophycin synthetases characterized so far, the purified enzyme from strain ADP1 did not accept lysine as an alternative substrate to arginine. The apparent -values for arginine (47 μM) and aspartic acid (240 μM) 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 values for ATP. The ATP-binding site responsible for the addition of arginine was found to have a much higher affinity for ATP (38 μM) 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-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 ADP1.

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2004-08-01
2019-11-12
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