Cyanophycinase
Asymmetric Unit of Cyanophycinase. PDB: 3EN0
Cyanophycinase consists of three identical chains.
Identifiers
EC no.3.4.15.6
CAS no.131554-16-0
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Biological assembly of cyanophycinase determined from the organism Synechocystis sp. PCC6803
The catalytic triad of Cyanophycinase is Ser 132, His 174, and Glu 201. Other conserved residues which form a pocket around the serine include: Gln101, Asp172, Gln173, Arg178, Arg180 and Arg183

Cyanophycinase (EC 3.4.15.6, cyanophycin degrading enzyme, beta-Asp-Arg hydrolysing enzyme, CGPase, CphB, CphE, cyanophycin granule polypeptidase, extracellular CGPase) is an enzyme.[1][2][3] It catalyses the following chemical reaction

[L-Asp(4-L-Arg)]n + H2O [L-Asp(4-L-Arg)]n-1 + L-Asp(4-L-Arg)

The enzyme is highly specific for the branched polypeptide cyanophycin. It is similar to Dipeptidase E, another S51 family serine protease.

Structure

The asymmetric unit of cyanophycinase consists of three identical chains, each containing 291 residues. The structure of cyanophycinase was determined from the freshwater cyanobacterium Synechocystis sp. PCC 6803 at 1.5-A resolution, which showed that the structure is dimeric.[4]

Enzyme function

Cyanophycinase is a carboxy terminal specific exopeptidase, meaning it catalyzes the cleavage of the carboxy terminal peptide bond of cyanophycin. It was hypothesized that cyanophycinase was a serine protease due to extreme inhibition of the enzyme when used with known serine protease inhibitors, such as DMSO. Site directed mutagenesis experiments confirmed that the enzyme is a serine protease and suggested that Ser 132 is the primary catalytic residue. Other key residues for specificity include Gln101, Asp172, Gln173, Arg178, Arg180 and Arg183 which form a conserved pocket adjacent to Ser 132. Kinetic characterization of the enzyme demonstrates that the enzyme displays Michaelis–Menten kinetics with a kcat of 16.5 s−1 and a kcat/KM of 7.5 × 106 M−1 s−1.[5]

Connection to nitrogen storage in Cyanobacteria

Cyanophycin is highly resistant to degradation by all conventional proteases, and the only enzyme known to be capable of hydrolyzing it is cyanophycinase. Cyanophycin is a non-ribosomally synthesized peptidyl polymer that is used for nitrogen storage by cyanobacteria and other select eubacteria. Approximately 90% of cyanobacteria are diazotrophic, meaning that they can grow without an external source of fixed nitrogen. Diazotrophic growth[6] was severely impaired in bacteria with a mutated cyanophycinase gene, indicating that the inability to degrade cyanophycin is detrimental for the diazotrophic growth of the cyanobacterium, due to an excess of nitrogen storage.

References

  1. ↑ Obst M, Krug A, Luftmann H, Steinbüchel A (July 2005). "Degradation of cyanophycin by Sedimentibacter hongkongensis strain KI and Citrobacter amalonaticus strain G Isolated from an anaerobic bacterial consortium". Applied and Environmental Microbiology. 71 (7): 3642–52. doi:10.1128/aem.71.7.3642-3652.2005. PMC 1169039. PMID 16000772.
  2. ↑ Obst M, Oppermann-Sanio FB, Luftmann H, Steinbüchel A (July 2002). "Isolation of cyanophycin-degrading bacteria, cloning and characterization of an extracellular cyanophycinase gene (cphE) from Pseudomonas anguilliseptica strain BI. The cphE gene from P. anguilliseptica BI encodes a cyanophycinhydrolyzing enzyme". The Journal of Biological Chemistry. 277 (28): 25096–105. doi:10.1074/jbc.m112267200. PMID 11986309.
  3. ↑ Richter R, Hejazi M, Kraft R, Ziegler K, Lockau W (July 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". European Journal of Biochemistry. 263 (1): 163–9. doi:10.1046/j.1432-1327.1999.00479.x. PMID 10429200.
  4. ↑ A.M. Law et al. "The structural basis of beta-peptide-specific cleavage by the serine protease cyanophycinase". J. Mol. Biol. (2009) https://doi.org/10.1016/j.jmb.2009.07.001
  5. ↑ A.M. Law et al. "The structural basis of beta-peptide-specific cleavage by the serine protease cyanophycinase". J. Mol. Biol. (2009) https://doi.org/10.1016/j.jmb.2009.07.001
  6. ↑ Picossi S, Valladares A, Flores E, Herrero A. "Nitrogen-regulated genes for the metabolism of cyanophycin, a bacterial nitrogen reserve polymer: expression and mutational analysis of two cyanophycin synthetase and cyanophycinase gene clusters in heterocyst-forming cyanobacterium Anabaena sp. PCC 7120" J Biol Chem. 2004 Mar 19;279(12):11582-92. doi: https://doi.org/10.1074/jbc.m311518200
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