Cyanase

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cyanase
Structure of the cyanase from E. coli with the di-anion oxalate bound at the enzyme active site
Identifiers
EC no.4.2.1.104
CAS no.37289-24-0
Databases
IntEnzIntEnz view
BRENDABRENDA entry
ExPASyNiceZyme view
KEGGKEGG entry
MetaCycmetabolic pathway
PRIAMprofile
PDB structuresRCSB PDB PDBe PDBsum
Gene OntologyAmiGO / QuickGO
Search
PMCarticles
PubMedarticles
NCBIproteins
Cyanate lyase, C-terminal domain
Identifiers
SymbolCyanate_lyase
PfamPF02560
InterProIPR003712
SCOP21dw9 / SCOPe / SUPFAM
CDDcd00559
Available protein structures:
Pfam  structures / ECOD  
PDBRCSB PDB; PDBe; PDBj
PDBsumstructure summary

The enzyme cyanase (EC 4.2.1.104, also known as cyanate hydratase or cyanate lyase), catalyses the bicarbonate dependent metabolism of cyanate to produce ammonia and carbon dioxide.[1][2] The systematic name of this enzyme is carbamate hydrolyase. In E. coli, cyanase is an inducible enzyme and is encoded for by the cynS gene.[3][2] Cyanate is a toxic anion, and cyanase catalyzes the metabolism into the benign products of carbon dioxide and ammonia.[1]

Enzyme Reaction[edit]

Cyanase catalyzes the metabolic conversion of toxic cyanate to carbamate (H2NCOO−). The enzyme does this by first catalyzing the conversion of cyanate to carbamate, which then spontaneously decomposes to carbon dioxide and ammonia:[4]

  1. cyanate (OCN−) + HCO3− + H+ carbamate (H2NCOO−) + CO2
  2. carbamate (H2NCOO−) + H+ NH3 + CO2 (spontaneous)

The resulting net reaction is:

cyanate (OCN−) + HCO3− + 2 H+ NH3 + 2 CO2
Cyanase enzyme catalyzes the reaction of bicarbonate and cyanate forming carbamate, to be rapidly decomposed into carbon dioxide and ammonia. The two adjacent anion binding sights signified by the catalytic Arg 96 residues on different monomers allow this reaction to occur in the pocket. Created on ChemDraw.

The kinetic mechanism is a rapid equilibrium where bicarbonate and cyanate both act as substrates. Bicarbonate acts a recycling substrate to cyanase in the production carbamate which then decomposes to complete the metabolism, rather than metabolism via a hydrolysis reaction as formerly believed.[5]

A positively charged active site forms a pocket with two anion binding sites, bonded via ionic interaction. Positively charged amino acids that make up the protein help form this site between subunits.[6][7] This adjacent anion binding site structure accommodates a complex of cyanate and bicarbonate, allowing the carbamate producing reaction to proceed.[7]

Enzyme Structure[edit]

Some bacteria can overcome the toxicity of environmental cyanate by degrading it via this enzyme.[8] Cyanate hydratase is found in bacteria and plants. Cyanase is functionally active as a homodecamer, composed of 5 complexes of dimers. Each of these monomer subunits is 17 kDa and supports half-site binding of substrates.[6] The cyanase monomer is composed of two domains. The N-terminal domain shows structural similarity to the DNA-binding alpha-helix bundle motif. The C-terminal domain has an 'open fold' with no structural homology to other proteins. The dimer structure reveals the C-terminal domains to be intertwined, and a decamer is formed by a pentamer of these dimers. The active site of the enzyme is located between dimers and is composed of residues from four adjacent subunits of the homodecamer.[6]

Connecting these two domains in the monomer is the single cysteine residue.[9] The disulfide bond links 156 amino acid units.[3] Forming the larger complexes of the pentamer and decamer requires the presence of cyanate, or a substrate analog like azide.[9] The final native enzyme complex is a complex of 5 dimers.[3]

The crystallization of cyanase shows that cyanase crystals are triclinic with one homodecamer in the asymmetric unit.[10]

The active site of cyanase accommodates two anions, bicarbonate and cyanate.[7] Three catalytic residues, Arg96, Glu99, and Ser122, form this pocket that stabilizes the anions with ionic interactions.[1] The positively charged and polar amino acid residues allow the active site to bind anions. The active site sits between dimers, noted by the Arg96 in each monomer to be in close proximity.[1]

Cyanase pentamer with labeled arginine residues shown. Circles indicate active pockets where the 96th residues are arginines and provide a catalytic pocket for the enzyme mechanism to take place. Created in PyMol from http://www.rcsb.org/structure/6B6M.

[11][10]

The structure of cyanase has been shown to be similar in its prokaryotic and eukaryotic forms.[12]

Enzyme Activity[edit]

Cyanase is regulated both transcriptionally and in its enzymatic activity.[13] Transcription of cyanase is elevated with the presence of extracellular cyanate, a toxic ion, but also down regulated by the presence of excess arginine, the catalytic amino acid.[13] Cyanase requires the product of the reaction catalyzed by cyanate permease.[2] Activity of cyanase is also dependent on the concentration of its substrates, cyanate and bicarbonate.[12]

Because bicarbonate is itself a recycling substrate and the kinetics of cyanase include a rapid and random equilibrium, bicarbonate can also act as an enzyme inhibitor.[7] At low concentrations, bicarbonate shows uncompetitive inhibition, where it binds to the one of enzyme's anionic binding sites, and inhibit cyanate binding, or bicarbonate can bind once more so that the enzyme complex is bound to two bicarbonates in its double anion active site.[6] At higher concentrations, the trend moves toward non-competitive inhibition, where the incorrect, dead-end complex must decompose to the initial separated units before the binding action can begin again.[7]

This random equilibrium of cyanate and bicarbonate complexing with the enzyme causing substrate inhibition is referred to as ping pong inhibition.[7]

Functional Relevance[edit]

Cyanase is an important enzyme for plants and microbacteria as cyanate is a toxic substance that is prevalent in many environments.[12] Further cyanase activity has been found to be encoded in cynH, a gene only found in marine cyanobacteria.[14] There is a dodecapeptide, a partial region near the N-terminus of cyanate, that as a novel cationic α-helical antimicrobial peptide, known by CL 14-25.[15]

Cyanase is also important as an energy metabolizer to fix Nitrogen in nitrifiers. Cyanate is an important source of reduced nitrogen in aquatic and terrestrial ecosystems, and although it is a toxic ion, it can be converted to ammonium and carbon dioxide by a cyanase enzyme, supplying energetic Nitrogen compounds to the organism.[16] The activity of cyanase is heavily dependent on the present of bicarbonate, and to overcome this bottleneck, a combined application of cyanase and carbonic anhydrase has been used in a study.[17][18]

References[edit]

  1. ^ a b c d Qian D, Jiang L, Lu L, Wei C, Li Y (March 2011). "Biochemical and structural properties of cyanases from Arabidopsis thaliana and Oryza sativa". PLOS ONE. 6 (3): e18300. Bibcode:2011PLoSO...618300Q. doi:10.1371/journal.pone.0018300. PMC 3070753. PMID 21494323.
  2. ^ a b c Anderson PM, Sung YC, Fuchs JA (December 1990). "The cyanase operon and cyanate metabolism". FEMS Microbiology Reviews. 7 (3–4): 247–52. doi:10.1111/j.1574-6968.1990.tb04920.x. PMID 2094285.
  3. ^ a b c Chin CC, Anderson PM, Wold F (January 1983). "The amino acid sequence of Escherichia coli cyanase" (PDF). The Journal of Biological Chemistry. 258 (1): 276–82. doi:10.1016/S0021-9258(18)33253-8. PMID 6336748.
  4. ^ "IUBMB Enzyme Nomenclature: EC 4.2.1.104".
  5. ^ Johnson WV, Anderson PM (July 1987). "Bicarbonate is a recycling substrate for cyanase". The Journal of Biological Chemistry. 262 (19): 9021–5. doi:10.1016/S0021-9258(18)48040-4. PMID 3110153.
  6. ^ a b c d Walsh MA, Otwinowski Z, Perrakis A, Anderson PM, Joachimiak A (May 2000). "Structure of cyanase reveals that a novel dimeric and decameric arrangement of subunits is required for formation of the enzyme active site". Structure. 8 (5): 505–14. doi:10.1016/S0969-2126(00)00134-9. PMC 3366510. PMID 10801492.
  7. ^ a b c d e f Anderson PM, Little RM (April 1986). "Kinetic properties of cyanase". Biochemistry. 25 (7): 1621–6. doi:10.1021/bi00355a026. PMID 3518792.
  8. ^ Sung YC, Fuchs JA (October 1988). "Characterization of the cyn operon in Escherichia coli K12". The Journal of Biological Chemistry. 263 (29): 14769–75. doi:10.1016/S0021-9258(18)68104-9. PMID 3049588.
  9. ^ a b Little RM, Anderson PM (July 1987). "Structural properties of cyanase. Denaturation, renaturation, and role of sulfhydryls and oligomeric structure in catalytic activity". The Journal of Biological Chemistry. 262 (21): 10120–6. doi:10.1016/S0021-9258(18)61086-5. PMID 3301828.
  10. ^ a b Butryn A, Stoehr G, Linke-Winnebeck C, Hopfner KP (April 2015). "Serendipitous crystallization and structure determination of cyanase (CynS) from Serratia proteamaculans". Acta Crystallographica Section F. 71 (Pt 4): 471–6. doi:10.1107/S2053230X15004902. PMC 4388186. PMID 25849512.
  11. ^ Xu, Y. (October 2, 2017). "Cyanase from Serratia proteamaculans". Worldwide Protein Data Bank (RCSB PDB). doi:10.2210/pdb6b6m/pdb.
  12. ^ a b c Schlachter CR, Klapper V, Wybouw N, Radford T, Van Leeuwen T, Grbic M, Chruszcz M (July 2017). "Structural Characterization of a Eukaryotic Cyanase from Tetranychus urticae". Journal of Agricultural and Food Chemistry. 65 (27): 5453–5462. doi:10.1021/acs.jafc.7b01333. PMID 28613863.
  13. ^ a b Elleuche S, Pöggeler S (November 2008). "A cyanase is transcriptionally regulated by arginine and involved in cyanate decomposition in Sordaria macrospora". Fungal Genetics and Biology. 45 (11): 1458–69. doi:10.1016/j.fgb.2008.08.005. PMID 18796334.
  14. ^ Kamennaya NA, Post AF (January 2011). "Characterization of cyanate metabolism in marine Synechococcus and Prochlorococcus spp" (PDF). Applied and Environmental Microbiology. 77 (1): 291–301. Bibcode:2011ApEnM..77..291K. doi:10.1128/AEM.01272-10. PMC 3019706. PMID 21057026.
  15. ^ Takei N, Takahashi N, Takayanagi T, Ikeda A, Hashimoto K, Takagi M, Hamada T, Saitoh E, Ochiai A, Tanaka T, Taniguchi M (April 2013). "Antimicrobial activity and mechanism of action of a novel cationic α-helical dodecapeptide, a partial sequence of cyanate lyase from rice". Peptides. 42: 55–62. doi:10.1016/j.peptides.2012.12.015. PMID 23270672. S2CID 56770.
  16. ^ Palatinszky M, Herbold C, Jehmlich N, Pogoda M, Han P, von Bergen M, Lagkouvardos I, Karst SM, Galushko A, Koch H, Berry D, Daims H, Wagner M (August 2015). "Cyanate as an energy source for nitrifiers". Nature. 524 (7563): 105–8. Bibcode:2015Natur.524..105P. doi:10.1038/nature14856. PMC 4539577. PMID 26222031.
  17. ^ Ranjan B, Pillai S, Permaul K, Singh S (April 2018). "A novel strategy for the efficient removal of toxic cyanate by the combinatorial use of recombinant enzymes immobilized on aminosilane modified magnetic nanoparticles". Bioresource Technology. 253: 105–111. doi:10.1016/j.biortech.2017.12.087. PMID 29331825.
  18. ^ Kebeish R, Al-Zoubi O (April 2017). "Expression of the cyanobacterial enzyme cyanase increases cyanate metabolism and cyanate tolerance in Arabidopsis". Environmental Science and Pollution Research International. 24 (12): 11825–11835. doi:10.1007/s11356-017-8866-z. PMID 28343358. S2CID 10098182.
This article incorporates text from the public domain Pfam and InterPro: IPR003712
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