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

Abstract

Most cyanobacteria use a single type of cyanophycin synthetase, CphA1, to synthesize the nitrogen-rich polymer cyanophycin. The genomes of many N 2 -fixing cyanobacteria contain an additional gene that encodes a second type of cyanophycin synthetase, CphA2. The potential function of this enzyme has been debated due to its reduced size and the lack of one of the two ATP-binding sites that are present in CphA1. Here, we analysed CphA2 from Anabaena variabilis ATCC 29413 and Cyanothece sp. PCC 7425. We found that CphA2 polymerized the dipeptide b-aspartyl-arginine to form cyanophycin. Thus, CphA2 represents a novel type of cyanophycin synthetase. A cphA2 disruption mutant of A. variabilis was generated. Growth of this mutant was impaired under high-light conditions and nitrogen deprivation, suggesting that CphA2 plays an important role in nitrogen metabolism under N 2 -fixing conditions. Electron micrographs revealed that the mutant had fewer cyanophycin granules, but no alteration in the distribution of granules in its cells was observed. Localization of CphA2 by immunogold electron microscopy demonstrated that the enzyme is attached to cyanophycin granules. Expression of CphA1 and CphA2 was examined in Anabaena WT and cphA mutant cells. Whilst the CphA1 level increased upon nitrogen deprivation, the CphA2 level remained nearly constant.

Cyanophycin and arginine metabolism in cyanobacteria

“…Because in Anabaena the genes in the cluster described in the previous paragraph (that is similar to the Synechocystis cluster) are responsible for more cyanophycin synthetase and cyanophycinase activities than the genes in the second cluster, the genes in the two clusters have been named chpA1/cphB1 and cphA2/cphB2, respectively [26]. The cphA2 gene has been found to encode a second type of cyanophycin synthetase that is widespread predominantly in N2-fixing cyanobacteria [27], in which the divergent arrangement of cphA2 and In those cyanobacteria that have CphA2, -aspartyl-arginine can be reincorporated into cyanophycin under conditions that have not yet been defined (Fig. 1A).…”

Microbiology Comment

“…Recycling CGP via CphA2 consumes less ATP than recycling via de novo synthesis from arginine and aspartate. However, if energy saving were the reason for the observed phenotype, as suggested by Klemke et al (2016), an even stronger impairment would be expected under low light conditions, where the cells are really energy-limited. As this is not the case, the observation of Klemke and colleagues points towards another, as yet unknown requirement for dynamic remodelling of CGP in filamentous cyanobacteria.…”

“…A deletion of the cphA2 gene resulted only in a minor loss of total CGP and thus CphA2 was considered to be of minor importance (Picossi et al, 2004). In the paper by Klemke et al (2016), the authors identified a distinct function for CphA2 that closes an unrecognized gap in cyanophycin metabolism. They demonstrate that CphA2 acts as a cyanophycin synthetase that uses the b-aspartyl-arginine dipeptide as substrate, consuming one molecule of ATP per reaction.…”

Cyanophycin: A Nitrogen-Rich Reserve Polymer

“…However, in many nitrogen-fixing cyanobacteria, an additional version of CphA is present, termed CphA2. In 2016, Klemke et al resolved the function of CphA2 [44]. Compared to CphA1, CphA2 has a reduced size and just one ATP-binding site.…”

“…A mutant lacking CphA2 shows only a minor decrease in the overall CGP content. However, a CphA2-deficient mutant displays similar defects under diazotrophic and high light conditions than a CphA1 mutant [15,44]. This observation suggests that the apparent "futile cycle" of CGP hydrolysis and immediate repolymerization is probably of physiological significance in the context of nitrogen fixation [17].…”