**4.1. Cyanophycin synthetase**

CGP is non-ribosomally synthesized from aspartate and arginine by cyanophycin synthetase (CphA1) (**Figure 2**). In 1976, CphA1 was enriched and characterized for the first time by Simion [35]. The enzyme incorporates aspartate and arginine in an elongation reaction, which requires ATP, KCl, MgCl2 and a sulfhydryl reagent (β-mercaptoethanol or DTT). For its activity, CphA1 needs a so far unknown CGP primer, as a starting point of the elongation reaction [35]. By using synthetically primers, Berg et al. could show that a single building block of CGP (β-Asp-Arg) does not serve as an efficient primer for CphA1 elongation reaction in vitro. The primers need to consist of at least three Asp-Arg building blocks (β-Asp-Arg)<sup>3</sup> to detect CphA1 activity [36]. Other peptides, like cell wall or other cellular components, have been suggested to serve as an alternative priming substance for the CphA1 reaction [37]. This could be an explanation for the functionality of CGP synthesis in recombinant bacteria, without the ability to produce native CGP primers [38]. Interestingly, the CphA1 of *Thermosynechococcus elongatus* strain BP-1 shows primer-independent CGP synthesis [39].

CGP was formally thought to be unique in cyanobacteria. In 2002, Krehenbrink et al. and Ziegler et al. discovered through evaluation of obligate heterotrophic bacteria genomes that many heterotrophic bacteria possess CGP synthetase genes [23, 24]. Genes of CGP metabolism occur in a

In 1971, Robert Simon isolated CGP granules for the first time by using differential centrifugation. Along with this study, CGP has shown its special and unique solubility behavior [26]. CGP is insoluble at physiological ionic strength and at neutral pH, but soluble in solutions which are acidic, basic or highly ionic. In non-ionic detergents such as Triton X-100, CGP is insoluble; however, in ionic detergents like SDS, it is soluble [6]. Present-day CGP extraction

The chemical structure of CGP was proposed in 1976 by Simon and Weathers [2]. According to this model, CGP has a polymer backbone consisting of α-linked aspartic acid residues. The α-amino group of arginine is linked via isopeptide bonds to the β-carboxylic group of every aspartyl moiety. Because every aspartate residue is linked to an arginine residue, CGP contains equimolar amounts of aspartate and arginine [2]. This structure has been confirmed via enzymatic degradation studies. CGP-degrading enzymes (see below) release β-Asp-Arg dipeptides [28]. CD spectroscopy data suggest that the acid-soluble and neutral insoluble forms of CGP have similar conformations. Both forms contain substantial fractions of β-pleated sheet structure [29]. Cyanobacterial CGP has a molecular weight and polydispersity ranging from 25 to 100 kDa [26]. In contrast, the native CGP producer *Acinetobacter* sp. ADP1 synthesizes CGP with a lower molecular weight ranging from 21 to 28 kDa [30]. Recombinant bacteria or genetically engineered yeast harboring heterologous expression of cyanobacterial CGP synthesis genes also show a lower molecular weight of 25–45 kDa [27, 31]. Transgenic plant-produced CGP also shows a reduced polydispersity between 20 and 35 kDa [32]. A possible explanation would be that cyanophycin synthesis in the native cyanobacterial background involves additional factors contributing the

wide range of different phylogenetic taxa and not closely related to cyanobacteria [25].

methods are based on its solubility at low pH and insolubility at neutral pH [27].

polymer length. These additional factors should also be absent in *Acinetobacter* sp. ADP1.

amount (higher than 31 mol%) is soluble at neutral pH [34].

**4. CGP metabolism**

**4.1. Cyanophycin synthetase**

Native CGP is exclusively composed of aspartate and arginine. By contrast, in CGP isolated from recombinant *E. coli* expressing cyanophycin synthetase (see below) from *Synechocystis* sp. PCC 6803, besides aspartate and arginine, lysine has been found [33]. The amount of incorporated lysine in CGP influences its solubility behavior. Recombinant CGP with a high lysine

CGP is non-ribosomally synthesized from aspartate and arginine by cyanophycin synthetase (CphA1) (**Figure 2**). In 1976, CphA1 was enriched and characterized for the first time by

**3. CGP characteristics**

88 Cyanobacteria

Today, CphA1 enzymes from several bacteria, including cyanobacteria and heterotrophic bacteria, have been purified and characterized [33, 39–42]. The molecular mass of the characterized CphA1 enzymes ranges from 90 to 130 kDa. The active form of CphA1s from *Synechocystis* sp. PCC6308 and *Anabaena variabilis* PCC7937 is most likely homodimeric [33, 41], while the primer-independent CphA1 from *Thermosynechococcus elongatus* strain BP-1 forms a homotetramer [39]. The primary structure of cyanobacterial CphA1 can be divided into two regions [33]. The C-terminal region shows sequence similarities to peptide ligases that include murein ligases and folyl poly-γ-glutamate ligase. The N-terminal part of CphA1 shows sequence similarities with another superfamily of ATP-dependent ligases that include carboxylate-thiol

**Figure 2.** Schematic illustration of CGP metabolism in cyanobacteria. CGP is synthesized from aspartate and arginine by CGP synthetase (CphA1) in an ATP-depending elongation reaction using CGP primers, containing of at least three Asp-Arg building blocks. Intracellular CGP degradation is catalyzed by the CGPase (CphB). The β-Asp-Arg dipeptides resulting from cleavage of CGP are further hydrolyzed by isoaspartyl dipeptidase, releasing aspartate and arginine. In many nitrogen-fixing cyanobacteria, an additional CGP synthetase is present, termed CphA2. CphA2 can use β-aspartylarginine dipeptides to resynthesize CGP.

and carboxylate-amine ligase. Since the C- and N-terminal parts show similarity to different superfamilies of ATP-dependent ligases, two ATP-binding sites and two different active sites have been predicted [36]. In vitro experiments revealed that arginine is probably bound in the C-terminal and aspartate in the N-terminal active site [43].

and studied in detail. CphB is a 29.4 kDa C-terminal exopeptidase, catalyzing the hydrolyzation of CGP to β-Asp-Arg dipeptides [28]. Based on sequence analysis and inhibitor sensitivity to serine protease inhibitors, CphB appears to be a serine-type exopeptidase related to dipeptidase E (PepE) [28]. According to its sequence, CphB contains a serine residue within a lipase box motive (Gly-Xaa-Ser-Xaa-Gly). The serine residue together with a glutamic acid residue and a histidine residue forms the catalytic triad, which is typical for serine-type peptidases [28]. In 2009, the crystal structure has been solved at a resolution of 1.5 Å, showing that CphB forms a dimer. Site-directed mutagenesis confirms that CphB is a serine-type peptidase, consisting of a conserved pocket with the catalytic Ser at position 132 [46]. Structure modeling indicates that the cleavage specificity occurs due to an extended conformation in the active site pocket. The unique conformation of the active site pocket requires β-linked aspartyl peptides for binding and catalysis, preventing CphB from non-specific cleavage of other polypeptides next to CGP [46].

Cyanophycin: A Nitrogen-Rich Reserve Polymer http://dx.doi.org/10.5772/intechopen.77049 91

In addition to CphB, which catalyzes the intracellular cleavage of CGP, other versions of cyanophycinase exist, catalyzing the extracellular hydrolysis of CGP. In 2002, Obst et al. isolated several Gram-negative bacteria from different habitats, which were able to utilize CGP as a source of carbon and energy [47, 48]. One isolate was affiliated as *Pseudomonas anguilliseptica* strain BI. In the supernatant of a *Pseudomonas anguilliseptica* culture, a cyanophycinase was found and purified, called CphE [47]. CphE exhibits a high specificity for CGP; however, proteins were not or only marginally hydrolyzed. Degradation products of CphE are β-Asp-Arg dipeptides. Inhibitor sensitivity studies indicated that the catalytic mechanism of CphE is related to serine-type proteases. CphE from *Pseudomonas anguilliseptica* strain BI exhibits an amino acid sequence identity 27–28% to intracellular CphB enzymes of cyanobacteria [47]. Today, extracellular CGPases has been found in a high variety of bacteria including Grampositive, Gram-negative, aerobic and anaerobic strains. This indicates that the extracellular cleavage and utilization of CGP as carbon, nitrogen and energy source is a common principle

In 2007, in silico analysis showed that CphB homologs are widely distributed in eubacteria, proposing eight different groups including intracellular and extracellular CGPases. CGPases from cyanobacteria belong to group I, II and partially group III (CphB1–3). Groups IV–VIII,

The last step in catabolism of CGP is the cleavage of β-Asp-Arg dipeptides to monomeric amino acids, arginine and aspartate (**Figure 2**). In 1999, Richter et al. found β-Asp-Arg dipeptides hydrolyzing activity in extracts of *Synechocystis* sp. PCC 6803 [28]. In *Synechocystis* sp. PCC 6803, the ORF sll0422 as well as ORF all3922 from *Anabaena* sp. PCC 7120 is annotated as "plant-type asparaginase," because of sequence similarities to the first cloned asparaginase from plants [54]. During characterization of plant-type asparaginase in general, including Sll0422 and All3922, Hejazi et al. were able to show that these enzymes are able to hydrolyze a wide range of isoaspartyl dipeptides [55]. Isoaspartyl peptides arise from two biological pathways: First, proteolytic degradation of modified proteins containing isoaspartyl residues and second, as primary degradation product of CGP cleavage from CGPases. Thus, the plant-type

including CphE, are present in a large variety of non-photosynthetic bacteria [25].

in nature [47–53].

**4.3. Aspartyl-arginine dipeptidase**

The mechanism of CGP synthesis by CphA1 has been suggested by Berg et al. in 2000, by measuring the step-wise incorporation of amino acids to the C-terminus of the CGP primer. The putative CGP elongation cycle starts at the C-terminal end of the poly-aspartate backbone. First, the carboxylic acid group of the poly-aspartate backbone is activated by transfer of the γ-phosphoryl group of ATP. In the second step, one aspartate is bound at the C-terminus of the growing polymer by its amino group, forming a peptide bound. Subsequently, the intermediate (β-Asp-Arg)n-Asp is transferred to the second active site of CphA1 and phosphorylated at the β-carboxyl group of the aspartate. Finally, the α-group of arginine is linked to the β-carboxyl group of aspartate, forming an isopeptide bound [36].

Various CphA1 enzymes have been characterized with respect to their substrate affinity and specificity. For CphA1 of *Synechocystis* sp. PCC 6308, apparent Km values were determined to be 450 μM for aspartate, 49 μM for arginine, 200 μM for ATP and 35 μg/ml CGP as priming substance. The lower Km of arginine compared to aspartate indicates a higher affinity of CphA1 towards arginine. During the in vitro reaction, CphA1 converts per mol incorporated amino acid 1.3 ± 0.1 mol ATP to ADP. The optimal reaction conditions of this enzyme were at pH 8.2 and 50°C [41].

CphA homologs are widely distributed in eubacteria. In silico analysis proposes 10 different groups of cyanophycin synthetases [25]. In cyanobacteria, cyanophycin synthetases of group I–III (CphA, CphA2 and CphA2') can be found.

Recently, the function of a cyanophycin synthetase of group II (CphA2) has been characterized. Most non-diazotrophic cyanobacteria use a single type of cyanophycin synthetase (CphA1). 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. CphA2 uses the product of CGP hydrolysis, β-aspartyl-arginine dipeptide as substrate to resynthesize cyanophycin, consuming one molecule of ATP per elongation. 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].

### **4.2. Cyanophycinase**

Since 1976, it is known that CGP is resistant against hydrolytic cleavage by several proteases or arginase [2, 45]. This resistance is probably due to the branched structure of CGP [38]. Therefore, the presence of a highly specified peptidase for CGP hydrolysis was suggested.

In 1999, Richter et al. reported a CGP hydrolyzing enzyme from the unicellular cyanobacterium *Synechocystis* sp. PCC 6803, called CphB [28] (**Figure 2**). During this study, CphB was purified and studied in detail. CphB is a 29.4 kDa C-terminal exopeptidase, catalyzing the hydrolyzation of CGP to β-Asp-Arg dipeptides [28]. Based on sequence analysis and inhibitor sensitivity to serine protease inhibitors, CphB appears to be a serine-type exopeptidase related to dipeptidase E (PepE) [28]. According to its sequence, CphB contains a serine residue within a lipase box motive (Gly-Xaa-Ser-Xaa-Gly). The serine residue together with a glutamic acid residue and a histidine residue forms the catalytic triad, which is typical for serine-type peptidases [28]. In 2009, the crystal structure has been solved at a resolution of 1.5 Å, showing that CphB forms a dimer. Site-directed mutagenesis confirms that CphB is a serine-type peptidase, consisting of a conserved pocket with the catalytic Ser at position 132 [46]. Structure modeling indicates that the cleavage specificity occurs due to an extended conformation in the active site pocket. The unique conformation of the active site pocket requires β-linked aspartyl peptides for binding and catalysis, preventing CphB from non-specific cleavage of other polypeptides next to CGP [46].

In addition to CphB, which catalyzes the intracellular cleavage of CGP, other versions of cyanophycinase exist, catalyzing the extracellular hydrolysis of CGP. In 2002, Obst et al. isolated several Gram-negative bacteria from different habitats, which were able to utilize CGP as a source of carbon and energy [47, 48]. One isolate was affiliated as *Pseudomonas anguilliseptica* strain BI. In the supernatant of a *Pseudomonas anguilliseptica* culture, a cyanophycinase was found and purified, called CphE [47]. CphE exhibits a high specificity for CGP; however, proteins were not or only marginally hydrolyzed. Degradation products of CphE are β-Asp-Arg dipeptides. Inhibitor sensitivity studies indicated that the catalytic mechanism of CphE is related to serine-type proteases. CphE from *Pseudomonas anguilliseptica* strain BI exhibits an amino acid sequence identity 27–28% to intracellular CphB enzymes of cyanobacteria [47]. Today, extracellular CGPases has been found in a high variety of bacteria including Grampositive, Gram-negative, aerobic and anaerobic strains. This indicates that the extracellular cleavage and utilization of CGP as carbon, nitrogen and energy source is a common principle in nature [47–53].

In 2007, in silico analysis showed that CphB homologs are widely distributed in eubacteria, proposing eight different groups including intracellular and extracellular CGPases. CGPases from cyanobacteria belong to group I, II and partially group III (CphB1–3). Groups IV–VIII, including CphE, are present in a large variety of non-photosynthetic bacteria [25].

### **4.3. Aspartyl-arginine dipeptidase**

and carboxylate-amine ligase. Since the C- and N-terminal parts show similarity to different superfamilies of ATP-dependent ligases, two ATP-binding sites and two different active sites have been predicted [36]. In vitro experiments revealed that arginine is probably bound in the

The mechanism of CGP synthesis by CphA1 has been suggested by Berg et al. in 2000, by measuring the step-wise incorporation of amino acids to the C-terminus of the CGP primer. The putative CGP elongation cycle starts at the C-terminal end of the poly-aspartate backbone. First, the carboxylic acid group of the poly-aspartate backbone is activated by transfer of the γ-phosphoryl group of ATP. In the second step, one aspartate is bound at the C-terminus of the growing polymer by its amino group, forming a peptide bound. Subsequently, the intermediate (β-Asp-Arg)n-Asp is transferred to the second active site of CphA1 and phosphorylated at the β-carboxyl group of the aspartate. Finally, the α-group of arginine is linked to the

Various CphA1 enzymes have been characterized with respect to their substrate affinity and specificity. For CphA1 of *Synechocystis* sp. PCC 6308, apparent Km values were determined to be 450 μM for aspartate, 49 μM for arginine, 200 μM for ATP and 35 μg/ml CGP as priming substance. The lower Km of arginine compared to aspartate indicates a higher affinity of CphA1 towards arginine. During the in vitro reaction, CphA1 converts per mol incorporated amino acid 1.3 ± 0.1 mol ATP to ADP. The optimal reaction conditions of this enzyme were at

CphA homologs are widely distributed in eubacteria. In silico analysis proposes 10 different groups of cyanophycin synthetases [25]. In cyanobacteria, cyanophycin synthetases of group

Recently, the function of a cyanophycin synthetase of group II (CphA2) has been characterized. Most non-diazotrophic cyanobacteria use a single type of cyanophycin synthetase (CphA1). 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. CphA2 uses the product of CGP hydrolysis, β-aspartyl-arginine dipeptide as substrate to resynthesize cyanophycin, consuming one molecule of ATP per elongation. 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].

Since 1976, it is known that CGP is resistant against hydrolytic cleavage by several proteases or arginase [2, 45]. This resistance is probably due to the branched structure of CGP [38]. Therefore, the presence of a highly specified peptidase for CGP hydrolysis was suggested.

In 1999, Richter et al. reported a CGP hydrolyzing enzyme from the unicellular cyanobacterium *Synechocystis* sp. PCC 6803, called CphB [28] (**Figure 2**). During this study, CphB was purified

C-terminal and aspartate in the N-terminal active site [43].

β-carboxyl group of aspartate, forming an isopeptide bound [36].

pH 8.2 and 50°C [41].

90 Cyanobacteria

**4.2. Cyanophycinase**

I–III (CphA, CphA2 and CphA2') can be found.

The last step in catabolism of CGP is the cleavage of β-Asp-Arg dipeptides to monomeric amino acids, arginine and aspartate (**Figure 2**). In 1999, Richter et al. found β-Asp-Arg dipeptides hydrolyzing activity in extracts of *Synechocystis* sp. PCC 6803 [28]. In *Synechocystis* sp. PCC 6803, the ORF sll0422 as well as ORF all3922 from *Anabaena* sp. PCC 7120 is annotated as "plant-type asparaginase," because of sequence similarities to the first cloned asparaginase from plants [54]. During characterization of plant-type asparaginase in general, including Sll0422 and All3922, Hejazi et al. were able to show that these enzymes are able to hydrolyze a wide range of isoaspartyl dipeptides [55]. Isoaspartyl peptides arise from two biological pathways: First, proteolytic degradation of modified proteins containing isoaspartyl residues and second, as primary degradation product of CGP cleavage from CGPases. Thus, the plant-type asparaginases, Sll0422 and All3922, have not only a function in asparagine catabolism but also in the final step of CGP and protein degradation [55].

heterocysts and vegetative cells. In the *cph1* operon, *cphB1* and *cphA1* were cotranscribed. In addition, *cphA1* can be expressed from independent promoters, of which one is constitutive and the other regulated by the global nitrogen control transcriptional factor NtcA [18].

In cluster *cph2*, the *cphB2* and *cphA2* genes were found in opposite orientation and both genes were expressed monocistronically. The genes were expressed under conditions of ammonia,

In addition to these two gene clusters, a third set of ORFs containing putative *cphA* and *cphB* genes was found in *Nostoc punctiforme* PCC 73102 and *Anabaena variabilis* ATCC 29413 [25].

Generally, CGP accumulation is triggered by cell growth arresting stress conditions, such as entry into stationary phase, light or temperature stress, limitation of macronutrients (with the exception of nitrogen starvation) or inhibition of translation by adding antibiotics like chloramphenicol [9, 10, 61]. All of these CGP triggering conditions result in a reduced or arrested growth. In exponential growth phase the amino acids arginine and aspartate are mostly used for protein biosynthesis with the consequence of a low intracellular level of free amino acids. Under growth-limiting conditions, protein biosynthesis is slowed down, which yields an excess of monomeric amino acids in the cytoplasm, triggering the CGP biosynthesis [10].

CGP accumulation also requires an excess of nitrogen. For the filamentous cyanobacterium *Calothrix* sp. strain PCC 7601, it was shown that CGP accumulation occurs preferably in the presence of ammonia [62]. The addition of amino acids to the media further increased CGP formation [63]. During process optimization studies for heterotrophic CGP production in the strain *Acinetobacter calcoaceticus* ADP1, it was shown that addition of arginine to the medium as sole carbon source increased CGP accumulation drastically. When, in *A. calcoaceticus* strain ADP1, CGP synthesis is induced by phosphate starvation, it accounts to 3.5% (w/w) of the cell dry matter (CDM) with ammonia as nitrogen source. Additional supply of the medium with arginine increases the CGP amount to 41.4% (w/w) (CDM). Notably, a combined supply of arginine and aspartate has a much lower stimulating effect to CGP accumulation than arginine alone [30]. A potential link between regulation of arginine biosynthesis and GCP metabolism was suggested in many previous studies. In a transposon mutagenesis study in the filamentous cyanobacterium *Nostoc ellipsosporum,* an arginine biosynthesis gene, *argL*, was interrupted by a transposon. This mutation partially impairs arginine biosynthesis but does not strictly result in l-arginine auxotrophy. Without arginine supplementation, heterocysts failed to fix nitrogen, akinetes were unable to germinate and CGP granules did not appear. However, when both nitrate and arginine are present in the media, the impaired arginine biosynthesis is bypassed. Under this condition, the mutant could form CGP and was able to differentiate

In metabolic engineering studies of the CGP production strain *Acinetobacter calcoaceticus* ADP1, several genes related to the arginine biosyntheses or its regulation were modified to yield higher amounts of arginine. As a consequence, significant higher CGP production was observed [65].

Generally, the expression of the *cph2* is lower compared to *cph1* [18].

**5.2. Dependence of CGP metabolism on arginine biosynthesis**

functional akinetes, which contained CGP granules [64].

supplementation, but the expression was higher in the absence of ammonia.

Cyanophycin: A Nitrogen-Rich Reserve Polymer http://dx.doi.org/10.5772/intechopen.77049 93

nitrate or N2

The mature isoaspartyl dipeptidases of *Synechocystis* sp. PCC 6803 and *Anabaena* sp. PCC 7120 consist of two protein subunits that are generated by autocleavage of the primary translation product between Gly-172 and Thr-173 (numbering according to *Synechocystis* sp. PCC 6803) within the conserved consensus sequence GT(I/V)G [55]. The native molecular weight of approximately 70kD of this enzyme suggests that it has a subunit structure of α<sup>2</sup> β2 (α derived from the N-terminal part and β from the C-terminal part of the precursor) [55].

In *Anabaena* sp. PCC 7120, all genes involved in CGP metabolism as well as the isoaspartyl dipeptidases All3922 are expressed in vegetative cells and heterocysts but in different expression levels. Both, CGP synthetases and CGPases are much higher expressed in heterocysts than in vegetative cells [56]. However, asparaginase All3922 is present in significantly lower levels in heterocysts than in vegetative cells [57]. A deletion of All3922 in *Anabaena* sp. PCC 7120 causes an increased accumulation of CGP and β-Asp-Arg dipeptides. Furthermore, a deletion mutant shows an impaired diazotrophic growth similar to the phenotype known from CphB deletion mutants in *Anabaena* sp. PCC 7120 [18, 57]. This observation implies that the first step of CGP catabolism, the cleavage catalyzed by CphB, takes place in the heterocyst. The released β-Asp-Arg dipeptides are transported to the adjacent vegetative cells. Isoaspartyl dipeptidase All3922, present in the vegetative cells, cleaves the β-Asp-Arg dipeptides and releases monomeric aspartate and arginine [57]. When CGP synthesis is not possible, due to a deletion of CphA, arginine and aspartate might be transferred directly from heterocysts. This explains the minor effects on diazotrophic growth in a CphA deletion mutant [15]. These results identified β-Asp-Arg dipeptides as nitrogen vehicle in diazotrophic heterocyst forming cyanobacteria, next to glutamine and arginine alone or with aspartate [57–59]. A benefit of β-Asp-Arg dipeptides as nitrogen transport substance is avoiding the release of free arginine and aspartate in the heterocyst. This indicates that CGP metabolism has evolved in multicellular heterocyst-forming cyanobacteria to increase the efficiency of nitrogen fixation [57].
