Cyanophycin Synthetase-Like Enzymes of Non-Cyanobacterial Eubacteria: Characterization of the Polymer Produced by a Recombinant Synthetase of Desulfitobacterium hafniense

Abstract

Some bacterial genomes were found to contain genes encoding putative proteins with considerable sequence homology to cyanophycin synthetase CphA of cyanobacteria. Such a gene from the Gram-positive, spore-forming anaerobe Desulfitobacterium hafniense was cloned. Expression in Escherichia coli resulted in the formation of a polydispers copolymer of aspartic acid and arginine, with a minor amount of lysine, of about 30 kDa molecular mass. In contrast to cyanophycin, this polymer was water-soluble. The structure of the polymer formed by the synthetase from Desulfitobacterium hafniense was studied by enzymatic degradation with the cyanophycin-specific hydrolase cyanophycinase, and by chemical and mass-spectroscopic analyses. Despite of the differences in solubility, indicating that both polymers cannot be completely identical, the chemical structure was found to be very similar to that of cyanophycin. The results suggest that the use of cyanophycin-like polymers as a nitrogen- rich reserve material is not restricted to cyanobacteria, and that such polymers may not necessarily be stored in granules.

Synthesis and Accumulation of Cyanophycin in Transgenic Strains of Saccharomyces cerevisiae

“…2A). This observation was surprising as the chemical structure of this form of CGP has been reported to be identical to the water-insoluble form (16,52); also, our analysis did not reveal any differences (data not shown). Presumably, relevant groups or regions in the CGP molecule that are recognized by the antibodies are disguised, resulting in an inability of the IgGs to bind to the soluble form.…”

“…The crude cell extract obtained after cell disruption was centrifuged for 10 min at 13,000 rpm at 4°C. The supernatant was used for the isolation of water-soluble CGP by applying a modified method described by Ziegler et al (52), using heat treatment, proteinase K digestion, and precipitation with 3 volumes of ethanol. After samples were subjected to proteinase K digestion, they were applied to Vivaspin 20 concentrators (Vivascience AG, Hannover, Germany), with a 10-kDa membrane to remove low-molecular-weight substances.…”

“…CGP is a polydispersed polymer; the molecular size distribution of CGP varies with the producing host strains (1,15,18,20,30,37,52). Due to its branched structure, CGP is not degradable by a wide range of proteinases (45).…”

“…CGP is insoluble at neutral pH and under physiological ionic strength, but it is soluble at low (Ͼ3) or high (Ͻ9) pH. Ziegler et al were the first to observe a water-soluble form of CGP after the heterologous expression of cphA from Desulfitobacterium hafniense strain DSM 10664 in Escherichia coli (52). A detailed study of the solubility behavior of CGP isolated from recombinant E. coli in inorganic salts has been carried out by Füser and Steinbüchel (16).…”

CphA2 is a novel type of cyanophycin synthetase in N2-fixing cyanobacteria

“…Notably, CphA1 from A. variabilis was not able to use b-aspartyl-arginine as substrate, failing to release phosphate or form cyanophycin under our experimental conditions in vitro. As a side note, CphA2 activity was enhanced in vitro by adding water-soluble cyanophycin from Desulfitobacterium hafniense (Otterbach, 2010;Ziegler et al, 2002) as an artificial primer (Fig. S1).…”