Cyanophycinase, a peptidase degrading the cyanobacterial reserve material multi‐L‐arginyl‐poly‐L‐aspartic acid (cyanophycin)

Richter, Ralf P.; Hejazi, Mahdi; Kraft, Regine; Ziegler, K.; Lockau, Wolfgang

doi:10.1046/j.1432-1327.1999.00479.x

Cited by 115 publications

(129 citation statements)

References 32 publications

(58 reference statements)

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“…Based on the observation that cphB and cphA from Synechocystis sp. PCC 6803 can independently be expressed in E. coli when cloned in the two possible orientations, it has been suggested that the cphB-cphA gene cluster would not be cotranscribed in this cyanobacterium (12). Additionally, cyanophycin synthetase and cyanophycinase of different cyanobacteria can show different relative abundances under different physiological conditions.…”

Section: Discussionmentioning

confidence: 99%

“…10, 12, and 37). Gene cphB1 would encode a cyanophycinase of 298 amino acids showing 99% identity to CphB from A. variabilis (12) and 60% identity in a 269-amino acid overlap to cyanophycinase from Synechocystis sp. PCC 6803 (37).…”

Section: Identification Of Two Clusters Of Cyanophycin Metabolismmentioning

confidence: 99%

“…Recently, the gene cphB encoding a cyanophycinase has been identified in the genomic sequence of Synechocystis sp. PCC 6803 (12), and ORFs 1 of both Synechocystis sp. and Anabaena sp.…”

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confidence: 99%

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Nitrogen-regulated Genes for the Metabolism of Cyanophycin, a Bacterial Nitrogen Reserve Polymer

Picossi

Valladares

Flores

et al. 2004

Journal of Biological Chemistry

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Two gene clusters each encoding the cyanophycinmetabolism enzymes cyanophycin synthetase and cyanophycinase are found in the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. In cluster cph1, the genes cphB1 and cphA1 were expressed in media containing ammonium, nitrate, or N 2 as nitrogen sources, but expression was higher in the absence of combined nitrogen taking place both in vegetative cells and heterocysts. Both genes were cotranscribed from three putative promoters located upstream of cphB1, and, additionally, the cphA1 gene was expressed monocistronically from at least two promoters located in the intergenic cphB1-cphA1 region. Both constitutive promoters and promoters dependent on the global nitrogen control transcriptional regulator NtcA were identified. In cluster cph2, the cphB2 and cphA2 genes, which are found in opposite orientations, were expressed as monocistronic messages in media containing ammonium, nitrate, or N 2 , but expression was higher in the absence of ammonium. Expression of the cph2 genes was lower than that of cph1 genes. Analysis of cph gene insertional mutants indicated that cluster cph1 genes contributed more than cluster cph2 genes to cyanophycin accumulation in the whole filament as well as in heterocysts. Diazotrophic growth was more severely impaired in cyanophycinase than in cyanophycin synthetase mutants, indicating that cyanophycin, although normally synthesized in the heterocysts, is not required for heterocyst function and that the inability to degrade this polymer is detrimental for the diazotrophic growth of the cyanobacterium.

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Section: Discussionmentioning

confidence: 99%

Section: Identification Of Two Clusters Of Cyanophycin Metabolismmentioning

confidence: 99%

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Nitrogen-regulated Genes for the Metabolism of Cyanophycin, a Bacterial Nitrogen Reserve Polymer

Picossi

Valladares

Flores

et al. 2004

Journal of Biological Chemistry

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“…The corresponding enzyme, cyanophycinase (CphB), was purified from a recombinant E. coli harboring the cphB gene from Synechocystis sp. PCC6803 and characterized in detail (8). Dipeptides consisting of arginine plus aspartic acid and free arginine were identified as products of CGP degradation in addition to small amounts of aspartic acid (8).…”

mentioning

confidence: 99%

“…PCC6803 and characterized in detail (8). Dipeptides consisting of arginine plus aspartic acid and free arginine were identified as products of CGP degradation in addition to small amounts of aspartic acid (8). In contrast to intracellular degradation, nothing is known about the extracellular decomposition of this biopolymer by bacteria or other microorganisms.…”

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confidence: 99%

Isolation of Cyanophycin-degrading Bacteria, Cloning and Characterization of an Extracellular Cyanophycinase Gene (cphE) from Pseudomonas anguilliseptica Strain BI

Obst¹,

Oppermann‐Sanio²,

Luftmann³

et al. 2002

Journal of Biological Chemistry

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Eleven bacteria capable of utilizing cyanophycin (cyanophycin granule polypeptide (CGP)) as a carbon source for growth were isolated. One isolate was taxonomically affiliated as Pseudomonas anguilliseptica strain BI, and the extracellular cyanophycinase (CphE) was studied because utilization of cyanophycin as a carbon source and extracellular cyanophycinases were hitherto not described. CphE was detected in supernatants of CGP cultures and purified from a corresponding culture of strain BI employing chromatography on the anion exchange matrix Q-Sepharose and on an arginineagarose affinity matrix. The mature form of the inducible enzyme consisted of one type of subunit with M r ‫؍‬ 43,000 and exhibited high specificity for CGP, whereas proteins and synthetic polyaspartic acid were not hydrolyzed or were only marginally hydrolyzed. Degradation products of the enzyme reaction were identified as aspartic acid-arginine dipeptides (␤-Asp-Arg) by high performance liquid chromatography and electrospray ionization mass spectrometry. The corresponding gene (cphE, 1254 base pairs) was identified in subclones of a cosmid gene library of strain BI by heterologous active expression in Escherichia coli, and its nucleotide sequence was determined. The enzyme exhibited only 27-28% amino acid sequence identity to intracellular cyanophycinases occurring in cyanobacteria. Analysis of the amino acid sequence of cphE revealed a putative catalytic triad consisting of the motif GXSXG plus a histidine and most probably a glutamate residue. In addition, the strong inhibition of the enzyme by Pefabloc ® and phenylmethylsulfonyl fluoride indicated that the catalytic mechanism of CphE is related to that of serine type proteases. Quantitative analysis on the release of ␤-Asp-Arg dipeptides from C-terminal labeled CGP gave evidence for an exo-degradation mechanism.Cyanophycin (cyanophycin granule polypeptide, CGP) 1 is a naturally occurring poly(amino acid), which is synthesized in most cyanobacteria as nitrogen, carbon, and energy storage compound in the early stationary growth phase (1, 2). The water-insoluble CGP is accumulated intracellularly in the form of membraneless granules (3) and is degraded by the cells when growth is resumed. The backbone of this unique biopolymer consists of ␣-amino-␣-carboxyl-linked L-aspartic acid monomers. Most of the ␤-carboxylic groups are covalently bound to the ␣-amino groups of L-arginine residues (4, 5); in recombinant Escherichia coli expressing cyanobacterial CGP-synthesizing enzymes (see below), a significant fraction of arginine is replaced by lysine (6).Although much information has been obtained concerning the non-ribosomal biosynthesis of CGP, which is catalyzed by the cyanophycin synthetase (CphA; see Ref. 7 and cited references therein), only a few reports are available on the intracellular degradation of CGP. Intracellular CGP degradation was first observed in crude extracts of soluble proteins prepared from cells of Anabaena cylindrica (5). The corresponding enzyme, cyanophycinase (CphB), was...

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Cyanophycin

Oppermann‐Sanio¹,

Steinbüchel²

2002

Biopolymers Online

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Introduction Historical Outline Chemical Structure Chemical Analysis and Detection Solubility Properties of CGP and Isolation Molecular Weight and Structure of CGP Quantification of CGP Variations in Composition and CGP‐like Polymers Occurrence Occurrence in Cyanobacteria Occurrence in Chemotrophic Bacteria Functions Physiology Biochemistry Primary Structures and Motifs Occurring in CGP Synthetases Assay of CGP Synthetase Activity and Substrate Specificity Mechanism of Catalysis Molecular Genetics Biodegradation Intracellular Mobilization Extracellular Breakdown Biotechnological Production of CGP Fermentative Production after Heterologous Expression of cphA in E. coli Isolation of CGP Fermentative Production after Heterologous Expression of cphA in other Bacteria In‐Vitro Biosynthesis of CGP Outlook and Perspectives Patents

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Cyanophycinase, a peptidase degrading the cyanobacterial reserve material multi‐L‐arginyl‐poly‐L‐aspartic acid (cyanophycin)

Cited by 115 publications

References 32 publications

Nitrogen-regulated Genes for the Metabolism of Cyanophycin, a Bacterial Nitrogen Reserve Polymer

Nitrogen-regulated Genes for the Metabolism of Cyanophycin, a Bacterial Nitrogen Reserve Polymer

Isolation of Cyanophycin-degrading Bacteria, Cloning and Characterization of an Extracellular Cyanophycinase Gene (cphE) from Pseudomonas anguilliseptica Strain BI

Cyanophycin

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