Identification and characterization of a carboxysomal γ-carbonic anhydrase from the cyanobacterium Nostoc sp. PCC 7120

Abstract

Carboxysomes are proteinaceous microcompartments that encapsulate carbonic anhydrase (CA) and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco); carboxysomes, therefore, catalyze reversible HCO3 dehydration and the subsequent fixation of CO2. The N- and C-terminal domains of the β-carboxysome scaffold protein CcmM participate in a network of protein–protein interactions that are essential for carboxysome biogenesis, organization, and function. The N-terminal domain of CcmM in the thermophile Thermosynechococcus elongatus BP-1 is also a catalytically active, redox regulated γ-CA. To experimentally determine if CcmM from a mesophilic cyanobacterium is active, we cloned, expressed and purified recombinant, full-length CcmM from Nostoc sp. PCC 7120 as well as the N-terminal 209 amino acid γ-CA-like domain. Both recombinant proteins displayed ethoxyzolamide-sensitive CA activity in mass spectrometric assays, as did the carboxysome-enriched TP fraction. NstCcmM209 was characterized as a moderately active and efficient γ-CA with a k cat of 2.0 × 104 s−1 and k cat/K m of 4.1 × 106 M−1 s−1 at 25 °C and pH 8, a pH optimum between 8 and 9.5 and a temperature optimum spanning 25–35 °C. NstCcmM209 also catalyzed the hydrolysis of the CO2 analog carbonyl sulfide. Circular dichroism and intrinsic tryptophan fluorescence analysis demonstrated that NstCcmM209 was progressively and irreversibly denatured above 50 °C. NstCcmM209 activity was inhibited by the reducing agent tris(hydroxymethyl)phosphine, an effect that was fully reversed by a molar excess of diamide, a thiol oxidizing agent, consistent with oxidative activation being a universal regulatory mechanism of CcmM orthologs. Immunogold electron microscopy and Western blot analysis of TP pellets indicated that Rubisco and CcmM co-localize and are concentrated in Nostoc sp. PCC 7120 carboxysomes.

This is a preview of subscription content, access via your institution.

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

References

  1. Agarwal R, Ortleb S, Sainis JK, Melzer M (2009) Immunoelectron microscopy for locating Calvin cycle enzymes in the thylakoids of Synechocystis 6803. Mol Plant 2:32–42

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  2. Alber BE, Ferry JG (1994) A carbonic anhydrase from the archaeon Methanosarcina thermophila. Proc Natl Acad Sci USA 91:6909–6913

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  3. Alber BE, Ferry JG (1996) Characterization of heterologously produced carbonic anhydrase from Methanosarcina thermophila. J Bacteriol 178:3270–3274

    CAS  PubMed Central  PubMed  Google Scholar 

  4. Alber BE, Colangelo CM, Dong J, Stålhandske CMV, Baird TT, Tu C, Fierke CA, Silverman DN, Scott RA, Ferry JG (1999) Kinetic and spectroscopic characterization of the gamma-carbonic anhydrase from the methanoarchaeon Methanosarcina thermophila. Biochem 38:13119–13128

    CAS  Article  Google Scholar 

  5. Berry S, Fischer JH, Kruip J, Hauser M, Wildner GF (2005) Monitoring cytosolic pH of carboxysome-deficient cells of Synechocystis sp PCC 6803 using fluorescence analysis. Plant Biol 7:342–347

    CAS  PubMed  Article  Google Scholar 

  6. Chen AH, Robinson-Mosher A, Savage DF, Silver PA, Polka JK (2013) The bacterial carbon-fixing organelle is formed by shell envelopment of preassembled cargo. PLoS One 8(9):e76127. doi:10.1371/journal.pone.0076127

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  7. Chengelis CP, Neal RA (1979) Hepatic carbonyl sulfide metabolism. Biochem Biophys Res Commun 90:993–999

    CAS  PubMed  Article  Google Scholar 

  8. Cot SSW, So AKC, Espie GS (2008) A multiprotein bicarbonate dehydration complex essential to carboxysome function in cyanobacteria. J Bacteriol 190:936–945

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  9. Del Prete S, Vullo D, De Luca V, Carginale V, Scozzafava A, Supuran CT, Capasso C (2013) A highly catalytically active γ-carbonic anhydrase from the pathogenic anaerobe Porphyromonas gingivalis and its inhibition profile with anions and small molecules. Bioorg Med Chem Lett 23:4067–4071

    PubMed  Article  Google Scholar 

  10. Espie GS, Kimber MS (2011) Carboxysomes: cyanobacterial RubisCO comes in small packages. Photosyn Res 109:7–20

    CAS  PubMed  Article  Google Scholar 

  11. Friedberg D, Jager KM, Kessel M, Silman NJ, Bergman B (1993) Rubisco but not Rubisco activase is clustered in the carboxysomes of the cyanobacterium Synechococcus sp. PCC 7942: mud-induced carboxysomeless mutants. Mol Microbiol 9:1193–1201

    CAS  PubMed  Article  Google Scholar 

  12. Fukuzawa H, Suzuki E, Komukai Y, Miyachi S (1992) A gene homologous to chloroplast carbonic anhydrase (icfA) is essential to photosynthetic carbon dioxide fixation by Synechococcus PCC7942. Proc Natl Acad Sci USA 89:4437–4441

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  13. Fulda S, Huang F, Nilsson F, Hagemann M, Norling B (2000) Proteomics of Synechocystis sp. strain PCC 6803: identification of periplasmic proteins in cells grown at low and high salt concentrations. Eur J Biochem 267:5900–5907

    CAS  PubMed  Article  Google Scholar 

  14. Greenfield NJ (2007) Using circular dichroism spectra to estimate protein secondary structure. Nat Protocols 1:2876–2890

    Article  Google Scholar 

  15. Hackenberg C, Huege J, Engelhardt A, Wittink F, Laue M, Matthijs HCP, Kopka J, Bauwe H, Hagemann M (2012) Low-carbon acclimation in carboxysome-less and photorespiratory mutants of the cyanobacterium Synechocystis sp. strain PCC 6803. Microbiology 158:398–413

    CAS  PubMed  Article  Google Scholar 

  16. Heinhorst S, Williams EB, Cai F, Murin CD, Shively JM, Cannon GC (2006) Characterization of the carboxysomal carbonic anhydrase CsoSCA from Halothiobacillus neapolitanus. J Bacteriol 188:8087–8094

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  17. Huang F, Hedman E, Funk C, Kieselbach T, Schroder WP, Norling B (2004) Isolation of outer membrane of Synechocystis sp PCC 6803 and its proteomic characterization. Mol Cell Proteomics 3:586–595

    CAS  PubMed  Article  Google Scholar 

  18. Innocenti A, Supuran CT (2010) Paraoxon, 4-nitrophenyl phosphate and acetate are substrates of α- but not of β-, γ- and ζ-carbonic anhydrases. Bioorg Med Chem Lett 20:6208–6212

    CAS  PubMed  Article  Google Scholar 

  19. Innocenti A, Zimmerman S, Ferry JG, Scozzafava A, Supuran CT (2004) Carbonic anhydrase inhibitors. Inhibition of the β-class enzyme from the methanoarchaeon Methanobacterium thermoautotrophicum (Cab) with anions. Bioorg Med Chem Lett 14:4563–4567

    CAS  PubMed  Article  Google Scholar 

  20. Jager KM, Bergman B (1990) Localization of a multifunctional chaperonin (GroELprotein) in nitrogen-fixing Anabaena PCC 7120: presence in vegetative cells and heterocysts. Planta 183:120–125

    Article  Google Scholar 

  21. Kelly SM, Jess TJ, Price NC (2005) How to study proteins by circular dichroism. Biochim Biophys Acta 1751:119–139

    CAS  PubMed  Article  Google Scholar 

  22. Khalifah RG (1971) Carbon dioxide hydration activity of carbonic anhydrase. J Biol Chem 246:2561–2573

    CAS  PubMed  Google Scholar 

  23. Kinney JN, Salmeen A, Cai F, Kerfeld CA (2012) Elucidating essential role of conserved carboxysomal protein CcmN reveals common feature of bacterial microcompartment assembly. J Biol Chem 287:17729–17736

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  24. Lichtle C, McKay RML, Gibbs SP (1992) Immunogold localization of Photosystem-I and Photosystem-II light-harvesting complexes in cryptomonad thylakoids. Biol Cell 74:187–194

    Article  Google Scholar 

  25. Long BM, Price GD, Badger MR (2005) Proteomic assessment of an established technique for carboxysome enrichment from Synechococcus PCC7942. Can J Bot 83:746–757

    CAS  Article  Google Scholar 

  26. Long BM, Badger MR, Whitney SM, Price GD (2007) Analysis of carboxysomes from Synechococcus PCC7942 reveals multiple rubisco complexes with carboxysomal proteins CcmM and CcaA. J Biol Chem 282:29323–29335

    CAS  PubMed  Article  Google Scholar 

  27. Long BM, Tucker L, Badger MR, Dean Price G (2010) Functional cyanobacterial β-carboxysomes have an absolute requirement for both long and short forms of the CcmM protein. Plant Physiol 153:285–293

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  28. Lorimer GH, Pierce J (1989) Carbonyl Sulfide -An alternate substrate for but not an activator of ribulose-1,5-bisphosphate carboxylase. J Biol Chem 264:2764–2772

    CAS  PubMed  Google Scholar 

  29. Ludwig M, Sültemeyer D, Price GD (2000) Isolation of ccmKLMN genes from the marine cyanobacterium, Synechococcus sp. PCC7002 (Cyanophyceae), and evidence that CcmM is essential for carboxysome assembly. J Phycol 36:1109–1118

    CAS  Article  Google Scholar 

  30. Marcus Y, Berry JA, Pierce J (1992) Photosynthesis and photorespiration in a mutant of the cyanobacterium Synechocystis PCC 6803 lacking carboxysomes. Planta 187:511–516

    CAS  PubMed  Article  Google Scholar 

  31. Mata-Cabana A, Florencio FJ, Lindahl M (2007) Membrane proteins from the cyanobacterium Synechocystis sp PCC 6803 interacting with thioredoxin. Proteomics 7:3953–3963

  32. McGinn PJ, Canvin DT, Coleman JR (1997) Influx and efflux of inorganic carbon during steady-state photosynthesis of air-grown Anabaena variabilis. Can J Bot 75:1913–1926

    CAS  Article  Google Scholar 

  33. McKay RML, Gibbs SP, Espie GS (1993) Effect of dissolved inorganic carbon on the expression of carboxysomes, localization of Rubisco and the mode of inorganic carbon transport in cells of the cyanobacterium Synechococcus UTEX 625. Arch Microbiol 159:21–29

    CAS  Article  Google Scholar 

  34. Miller AG, Espie GS, Canvin DT (1989) Use of carbon oxysulphide, a structural analog of CO2, to study active CO2 transport in the cyanobacterium Synechococcus UTEX 625. Plant Physiol 90:1221–1231

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  35. Miller AG, Salon C, Espie GS, Canvin DT (1997) Measurement of the amount and isotopic composition of the CO2 released from the cyanobacterium Synechococcus UTEX 625 after rapid quenching of the active CO2 transport system. Can J Bot 75:981–997

    CAS  Article  Google Scholar 

  36. Moslavac S, Bredemeier R, Mirus O, Granvogl B, Eichacker LA, Schleiff E (2005) Proteomic analysis of the outer membrane of Anabaena sp strain PCC 7120. J Proteome Res 4:1330–1338

    CAS  PubMed  Article  Google Scholar 

  37. Orus MI, Rodriguez-Buey ML, Marco E, Fernandez-Valiente E (2001) Changes in carboxysome structure and grouping and in photosynthetic affinity for inorganic carbon in Anabaena strain PCC 7119 (cyanophyta) in response to modification of CO2 and Na+ supply. Plant Cell Physiol 42:46–53

    CAS  PubMed  Article  Google Scholar 

  38. Peña KL, Castel SE, De Araujo C, Espie GS, Kimber MS (2010) Structural basis of the oxidative activation of the carboxysomal γ-carbonic anhydrase, CcmM. Proc Natl Acad Sci USA 107:2455–2460

    PubMed Central  PubMed  Article  Google Scholar 

  39. Pocker Y, Sarkanen S (1978) Carbonic anhydrase: structure catalytic versatility, and inhibition. Adv Enzymol Relat Areas Mol Biol 47:149–274

    CAS  PubMed  Google Scholar 

  40. Price GD, Badger MR (1989) Isolation and characterization of high CO2-requiring-mutants of the cyanobacterium Synechococcus PCC 7942–2 phenotypes that accumulate inorganic carbon but are apparently unable to generate CO2 within the carboxysome. Plant Physiol 91:514–525

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  41. Price GD, Badger MR, Woodger FJ, Long BM (2008) Advances in understanding the cyanobacterial CO2-concentrating- mechanism (CCM): functional components, Ci transporters, diversity, genetic regulation and prospects for engineering into plants. J Expt Bot 59:1441–1461

    CAS  Article  Google Scholar 

  42. Protoschill-Krebs G, Kesselmeier J (1992) Enzymatic pathways for the comsumption of carbonyl sulphide (COS) by higher plants. Bot Acta 105:206–212

    CAS  Article  Google Scholar 

  43. Rae BD, Long BM, Badger MR, Price GD (2013) Functions, compositions, and evolution of the two types of carboxysomes: polyhedral microcompartments that facilitate CO2 fixation in cyanobacteria and some proteobacteria. Microbiol Mol Biol Rev 77:357–379

    CAS  PubMed  Article  Google Scholar 

  44. Rowlett RS, Gargiulo NJ, Santoli FA, Jackson JM, Corbett AH (1991) Activation and inhibition of bovine carbonic anhydrase-III by dianions. J Biol Chem 266:933–941

    CAS  PubMed  Google Scholar 

  45. Rowlett RS, Tu C, McKay MM, Preiss JR, Loomis RJ, Hicks KA, Marchione RJ, Strong JA, Donovan GS, Chamberlin JE (2002) Kinetic characterization of wild-type and proton transfer-impaired variants of β-carbonic anhydrase from Arabidopsis thaliana. Arch Biochem Biophys 404:197–209

    CAS  PubMed  Article  Google Scholar 

  46. Sawaya MR, Cannon GC, Heinhorst S, Tanaka S, Williams EB, Yeates TO, Kerfeld CA (2006) The structure of β-carbonic anhydrase from the carboxysomal shell reveals a distinct subclass with one active site for the price of two. J Biol Chem 281:7546–7555

    CAS  PubMed  Article  Google Scholar 

  47. Silverman DN (1982) Carbonic anhydrase: oxygen-18 exchange catalyzed by an enzyme with rate-contributing proton-transfer steps. Methods Enzymol 87:732–753

    CAS  PubMed  Article  Google Scholar 

  48. So AKC, Espie GS (2005) Cyanobacterial carbonic anhydrases. Can J Bot 83:721–734

    CAS  Article  Google Scholar 

  49. So AKC, Van Spall HGC, Coleman JR, Espie GS (1998) Catalytic exchange of 18O from 13C18O-labelled CO2 by wild-type cells and ecaA, ecaB, and ccaA mutants of the cyanobacteria Synechococcus PCC7942 and Synechocystis PCC6803. Can J Bot 76:1153–1160

    CAS  Google Scholar 

  50. So AKC, Cot SSW, Espie GS (2002a) Characterization of the C-terminal extension of carboxysomal carbonic anhydrase from Synechocystis sp. PCC6803. Funct Plant Biol 29:183–194

    CAS  Article  Google Scholar 

  51. So AKC, John-McKay M, Espie GS (2002b) Characterization of a mutant lacking carboxysomal carbonic anhydrase from the cyanobacterium Synechocystis PCC6803. Planta 214:456–467

    CAS  PubMed  Article  Google Scholar 

  52. So AKC, Espie GS, Williams EB, Shively JM, Heinhorst S, Cannon GC (2004) A novel evolutionary lineage of carbonic anhydrase (ε Class) is a component of the carboxysome shell. J Bacteriol 186:623–630

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  53. Soltes-Rak E, Mulligan ME, Coleman JR (1997) Identification and characterization of a gene encoding a vertebrate α- type carbonic anhydrase in cyanobacteria. J Bacteriol 179:769–774

    CAS  PubMed Central  PubMed  Google Scholar 

  54. Tripp BC, Ferry JG (2000) A structure-function study of a proton transport pathway in the gamma-class carbonic anhydrase from Methanosarcina thermophila. Biochemistry 39:9232–9240

    CAS  PubMed  Article  Google Scholar 

  55. Wilbur KM, Anderson NG (1948) Electrometric and colorimetric determination of carbonic anhydrase. J Biol Chem 176:147–154

    CAS  PubMed  Google Scholar 

  56. Yu JW, Price GD, Song L, Badger MR (1992) Isolation of a putative carboxysomal carbonic anhydrase gene from the cyanobacterium Synechococcus PCC7942. Plant Physiol 100:794–800

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  57. Zimmerman SA, Tomb JF, Ferry JG (2010) Characterization of CamH from Methanosarcina thermophila, founding member of a subclass of the γ class of carbonic anhydrases. J Bacteriol 192:1353–1360

    CAS  PubMed Central  PubMed  Article  Google Scholar 

Download references

Acknowledgments

This study was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) to G. S. E. and M. S. K. and an NSERC CGSD to C. A.; by a grant from the Australian Research Council to B. M. L. and G. D. P., and by a grant from the U.S.A. National Science Foundation, MCB-1157332, to R. S. R.

Author information

Affiliations

Authors

Corresponding author

Correspondence to George S. Espie.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 27 kb)

Fig. S1

Immunogold localization of Rubisco and CcmM in Nostoc PCC 7120. Typical sections of Nostoc PCC 7120 immunolabeled with a, b anti-Rubsico (RbcL/S) antibody and c, d anti-CcmM CT antibody. Protein A coated gold particles (15 nm) were used to visualize the immune-complexes. Carboxysomes are indicated by c. Scale bars represent a, b, d, 100 nm or c 500 nm

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

de Araujo, C., Arefeen, D., Tadesse, Y. et al. Identification and characterization of a carboxysomal γ-carbonic anhydrase from the cyanobacterium Nostoc sp. PCC 7120. Photosynth Res 121, 135–150 (2014). https://doi.org/10.1007/s11120-014-0018-4

Download citation

Keywords

  • Carboxysome
  • CcmM
  • CO2 concentrating mechanism
  • Carbonyl sulfide
  • Gamma-carbonic anhydrase
  • Nostoc sp. PCC 7120