A cyanophycin synthetase from Thermosynechococcus elongatus BP-1 catalyzes primer-independent cyanophycin synthesis.
Cyanophycin synthesis is catalyzed by cyanophycin synthetase (CphA). It was believed that CphA requires L-aspartic acid (Asp), L-arginine (Arg), ATP, Mg2+, and a primer (low-molecular mass cyanophycin) for cyanophycin synthesis and catalyzes the elongation of a low-molecular mass cyanophycin. Despite extensive studies of cyanophycin, the mechanism of primer supply is still unclear, and already-known CphAs were primer-dependent enzymes. In the present study, we found that recombinant CphA from Thermosynechococcus elongatus BP-1 (Tlr2170 protein) catalyzed in vitro cyanophycin synthesis in the absence of a primer. The Tlr2170 protein showed strict substrate specificity toward Asp and Arg. The optimum pH was 9.0, and Mg2+ or Mn2+ was essential for cyanophycin synthesis. KCl enhanced the cyanophycin synthesis activity of the Tlr2170 protein; in contrast, dithiothreitol did not. The Tlr2170 protein appeared to be a 400+/-9 kDa homo-tetramer. The Tlr2170 protein showed thermal stability and retained its 80% activity after a 60-min incubation at 50 degrees C. In addition, we examined cyanophycin synthesis at 30 degrees C, 40 degrees C, 50 degrees C, and 60 degrees C. SDS-PAGE analysis showed that the molecular mass of cyanophycin increased with increased reaction temperature.
Arai
Toshinobu
T
Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo, 169-8555, Japan.
Kino
Kuniki
K
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000458:Cyanobacteria / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D008969:Molecular Sequence Data; D010453:Peptide Synthases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010802:Phylogeny; D010940:Plant Proteins / Q000378:metabolism*; D013379:Substrate Specificity
D001426:Bacterial Proteins; D010940:Plant Proteins; C024250:cyanophycin; D010453:Peptide Synthases; C113257:cyanophycin synthase, bacteria