Skip to main content
SpringerLink
Log in

A cyanophycin synthetase from Thermosynechococcus elongatus BP-1 catalyzes primer-independent cyanophycin synthesis

  • Biotechnologically Relevant Enzymes and Proteins
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

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°C. In addition, we examined cyanophycin synthesis at 30°C, 40°C, 50°C, and 60°C. SDS-PAGE analysis showed that the molecular mass of cyanophycin increased with increased reaction temperature.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Aboulmagd E, Oppermann-Sanio FB, Steinbüchel A (2000) Molecular characterization of the cyanophycin synthetase from Synechocystis sp. strain PCC6308. Arch Microbiol 174:297–306

    Article  CAS  Google Scholar 

  • Aboulmagd E, Oppermann-Sanio FB, Steinbüchel A (2001) Purification of Synechocystis sp. strain PCC6308 cyanophycin synthetase and its characterization with respect to substrate and primer specificity. Appl Environ Microbiol 67:2176–2182

    CAS  PubMed  PubMed Central  Google Scholar 

  • Berg H, Zeigler K, Piotukh K, Baier K, Lockau W, Volkmer-Engert R (2000) Biosynthesis of the cyanobacterial reserve polymer multi-l-arginyl-poly-l-aspartic acid (cyanophycin): mechanism of the cyanophycin synthetase reaction studied with synthetic primers. Eur J Biochem 267:5561–5570

    Article  CAS  Google Scholar 

  • Fan C, Moews PC, Shi Y, Walsh CT, Knox JR (1994) Vancomycin resistance: structure of d-alanine: d-alanine ligase at 2.3 Å resolution. Science 266:439–443

    Article  CAS  Google Scholar 

  • Fan C, Moews PC, Shi Y, Walsh CT, Knox JR (1995) A common fold for peptide synthetases cleaving ATP to ADP: glutathione synthetase and d-alanine:d-alanine ligase of Escherichia coli. Proc Natl Acad Sci U S A 92:1172–1176

    Article  CAS  Google Scholar 

  • Frey KM, Oppermann-Sanio FB, Schmidt H, Steinbüchel A (2002) Technical-scale production of cyanophycin with recombinant strains of Escherichia coli. Appl Environ Microbiol 68:3377–3384

    Article  CAS  Google Scholar 

  • Füser G, Steinbüchel A (2007) Analysis of genome sequences for genes of cyanophycin metabolism: identifying putative cyanophycin metabolizing prokaryotes. Macromol Biosci 7:278–296

    Article  Google Scholar 

  • Gyenes T, Torma V, Gyarmati B, Zrínyi M (2008) Synthesis and swelling properties of novel pH-sensitive poly(aspartic acid) gels. Acta Biomater 4:733–744

    Article  CAS  Google Scholar 

  • Hai TFB, Oppermann-Sanio FB, Steinbüchel A (1999) Purification and characterization of cyanophycin and cyanophycin synthetase from the thermophilic Synechococcus sp. MA19. FEMS Microbiol Lett 181:229–236

    Article  CAS  Google Scholar 

  • Hai T, Oppermann-Sanio FB, Steinbüchel A (2000) Molecular characterization of a thermostable cyanophycin synthetase from the thermophilic cyanobacterium Synechococcus sp. strain MA19 and in vitro synthesis of cyanophycin and related polyamides. Appl Environ Microbiol 68:93–101

    Article  Google Scholar 

  • Kino K, Kuratsu S, Noguchi A, Kokubo M, Nakazawa Y, Arai T, Yagasaki M, Kirimura K (2007) Novel substrate specificity of glutathione synthesis enzymes from Streptococcus agalactiae and Clostridium acetobutylicum. Biochem Biophys Res Commun 352:351–359

    Article  CAS  Google Scholar 

  • Kino K, Nakazawa Y, Yagasaki M (2008a) Dipeptide synthesis by l-amino acid ligase from Ralstonia solanacearum. Biochem Biophys Res Commun 371:536–540

    Article  CAS  Google Scholar 

  • Kino K, Noguchi A, Yagasaki M (2008b) Novel l-amino acid α-ligase from Bacillus licheniformis. J Biosci Bioeng (in press)

  • Krehenbrink M, Oppermann-Sanio FB, Steinbüchel A (2002) Evaluation of non-cyanobacterial genome sequences for occurrence of genes encoding proteins homologous to cyanophycin synthetase and cloning of an active cyanophycin synthetase from Acinetobacter sp. strain DSM 587. Arch Microbiol 177:371–380

    Article  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  Google Scholar 

  • Mooibroek H, Oosterhuis N, Giuseppin M, Toonen M, Franssen H, Scott E, Sanders J, Steinbüchel A (2007) Assessment of technological options and economical feasibility for cyanophycin biopolymer and high-value amino acid production. Appl Microbiol Biotechnol 77:257–267

    Article  CAS  Google Scholar 

  • Murzin AG (1996) Structural classification of proteins: new superfamilies. Curr Opin Struct Biol 6:386–394

    Article  CAS  Google Scholar 

  • Neumann K, Stephan DP, Ziegler K, Hühns M, Broer I, Lockau W, Pistorius EK (2005) Production of cyanophycin, a suitable source for the biodegradable polymer polyaspartate, in transgenic plants. Plant Biotechnol J 3:249–258

    Article  CAS  Google Scholar 

  • Richter R, Hejazi M, Kraft R, Ziegler K, Lockau W (1999) Cyanophycinase, a peptidase degrading the cyanobacterial reserve material multi-l-arginyl-poly-l-aspartic acid (cyanophycin): molecular cloning of the gene of Synechocystis sp. PCC 6803, expression in Escherichia coli, and biochemical characterization of the purified enzyme. Eur J Biochem 263:163–169

    Article  CAS  Google Scholar 

  • Sambrook J, Russell WD (2001) Molecular cloning: a laboratory manual,, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

    Google Scholar 

  • Sato M, Kirimura K, Kino K (2005) d-Amino acid dipeptides production utilizing d-alanine-d-alanine ligases with novel substrate specificity. J Biosci Bioeng 99:623–628

    Article  CAS  Google Scholar 

  • Schwamborn M (1998) Chemical synthesis of polyaspartates: a biodegradable alternative to currently used polycarboxylate homo- and copolymers. Polym Degrad Stab 89:39–45

    Article  Google Scholar 

  • Simon RD (1971) Cyanophycin granules from the blue-green alga Anabaena cylindrica: a reserve material consisting of copolymers of aspartic acid and arginine. Proc Natl Acad Sci U S A 68:265–267

    Article  CAS  Google Scholar 

  • Simon RD (1976) The biosynthesis of multi-l-arginyl-poly(l-aspartic acid) in the filamentous cyanobacterium Anabaena cylindrica. Biochim Biophys Acta 422:407–418

    Article  CAS  Google Scholar 

  • Simon RD, Weathers P (1976) Determination of the structure of the novel polypeptide containing aspartic acid and arginine which is found in cyanobacteria. Biochim Biophys Acta 420:165–176

    Article  CAS  Google Scholar 

  • Stubbe J, Tian J, He A, Sinskey AJ, Lawrence AG, Liu P (2005) Nontemplate-dependent polymerization processes: polyhydroxyalkanoate synthases as a paradigm. Annu Rev Biochem 74:433–480

    Article  CAS  Google Scholar 

  • Tabata K, Ikeda H, Hashimoto S (2005) ywfE in Bacillus subtilis codes for a novel enzyme, l-amino acid ligase. J Bacteriol 187:5195–5202

    Article  CAS  Google Scholar 

  • Tabata K, Hashimoto S (2007) Fermentative production of l-alanyl-l-glutamine by a metabolically engineered Escherichia coli strain expressing l-amino acid alpha-ligase. Appl Environ Microbiol 73:6378–6385

    Article  CAS  Google Scholar 

  • Voss I, Diniz SC, Aboulmagd E, Steinbüchel A (2004) Identification of the Anabaena sp. strain PCC7120 cyanophycin synthetase as suitable enzyme for production of cyanophycin in gram-negative bacteria like Pseudomonas putida and Ralstonia eutropha. Biomacromolecules 5:1588–1595

    Article  CAS  Google Scholar 

  • Yamamoto T, Shiraki K, Fujiwara S, Takagi M, Fukui K, Imanaka T (1999) In vitro heat effect on functional and conformational changes of cyclodextrin glucanotransferase from hyperthermophilic archaea. Biochem Biophys Res Commun 265:57–61

    Article  CAS  Google Scholar 

  • Ziegler K, Diener A, Herpin C, Richter R, Deutzmann R, Lockau W (1998) Molecular characterization of cyanophycin synthetase, the enzyme catalyzing the biosynthesis of the cyanobacterial reserve material multi-l-arginyl-poly-l-aspartate (cyanophycin). Eur J Biochem 254:154–159

    Article  CAS  Google Scholar 

  • Ziegler K, Deutzmann R, Lockau W (2002) Cyanophycin synthetase-like enzymes of non-cyanobacterial eubacteria: characterization of the polymer produced by a recombinant synthetase of Desulfitobacterium hafniense. Z Naturforsch 57c:522–529

    Article  Google Scholar 

Download references

Acknowledgments

We thank Kazusa DNA Research Institute (Chiba, Japan) for supplying the genomic DNA of T. elongatus BP-1 and Synechocystis sp. PCC6803. This work is financially supported in part by the Global COE program of the MEXT “Center for Practical Chemical Wisdom” and in part by a Waseda University Grant for Special Research Project “2008B-186”.

Author information

Authors and Affiliations

  1. Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo, 169-8555, Japan

    Toshinobu Arai & Kuniki Kino

Authors
  1. Toshinobu Arai
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kuniki Kino
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kuniki Kino.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Arai, T., Kino, K. A cyanophycin synthetase from Thermosynechococcus elongatus BP-1 catalyzes primer-independent cyanophycin synthesis. Appl Microbiol Biotechnol 81, 69–78 (2008). https://doi.org/10.1007/s00253-008-1623-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-008-1623-y

Keywords

Search

Navigation