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Volume 164, Issue 4

Putative extracellular α-class carbonic anhydrase, EcaA, of PCC 7942 is an active enzyme: a sequel to an old story Free

Abstract

Carbonic anhydrase (CA) EcaA of PCC 7942 was previously characterized as a putative extracellular α-class CA, however, its activity was never verified. Here we show that EcaA possesses specific CA activity, which is inhibited by ethoxyzolamide. An active EcaA was expressed in heterologous bacterial system, which supports the formation of disulfide bonds, as a full-length protein (EcaA+L) and as a mature protein that lacks a leader peptide (EcaA-L). EcaA-L exhibited higher specific activity compared to EcaA+L. The recombinant EcaA, expressed in a bacterial system that does not support optimal disulfide bond formation, exhibited extremely low activity. This activity, however, could be enhanced by the thiol-oxidizing agent, diamide; while a disulfide bond-reducing agent, dithiothreitol, further inactivated the enzyme. Intact cells that overexpress EcaA+L possess a small amount of processed protein, EcaA-L, whereas the bulk of the full-length protein resides in the cytosol. This may indicate poor recognition of the EcaA leader peptide by protein export systems. possessed a relatively low level of mRNA, which varied insignificantly in response to changes in CO supply. However, the presence of protein in the cells is not obvious. This points to the physiological insignificance of EcaA in , at least under the applied experimental conditions.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): alpha-class carbonic anhydrase , cyanobacteria , EcaA and Synechococcus elongatus PCC 7942
© 2018 The Authors
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2018-04-01
2024-04-23
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References

  1. Supuran CT. Structure and function of carbonic anhydrases. Biochem J 2016; 473:2023–2032 [View Article][PubMed]
     [Google Scholar]
  2. Smith KS, Ferry JG. Prokaryotic carbonic anhydrases. FEMS Microbiol Rev 2000; 24:335–366 [View Article][PubMed]
     [Google Scholar]
  3. So AKC, Espie GS. Cyanobacterial carbonic anhydrases. Can J Bot 2005; 83:721–734 [Crossref]
     [Google Scholar]
  4. Kupriyanova E, Pronina N, Los D. Carbonic anhydrase — a universal enzyme of the carbon-based life. Photosynthetica 2017; 55:3–19 [View Article]
     [Google Scholar]
  5. Price GD, Badger MR, Woodger FJ, Long BM. Advances in understanding the cyanobacterial CO2-concentrating mechanism (CCM): functional components, Ci transporters, diversity, genetic regulation and prospects for engineering into plants. J Exp Bot 2008; 59:1441–1461 [View Article][PubMed]
     [Google Scholar]
  6. Cannon GC, Heinhorst S, Kerfeld CA. Carboxysomal carbonic anhydrases: Structure and role in microbial CO2 fixation. Biochim Biophys Acta 2010; 1804:382–392 [View Article][PubMed]
     [Google Scholar]
  7. Kimber MS. Carboxysomal carbonic anhydrases. In Frost SC, McKenna R. (editors) Carbonic Anhydrase: Mechanism, Regulation, Links to Disease, and Industrial Applications New York, London: Dordrecht Heidelberg Springer; 2014 pp. 89–103 [Crossref]
     [Google Scholar]
  8. Soltes-Rak E, Mulligan ME, Coleman JR. Identification and characterization of a gene encoding a vertebrate-type carbonic anhydrase in cyanobacteria. J Bacteriol 1997; 179:769–774 [View Article][PubMed]
     [Google Scholar]
  9. So АK, van Spall НGC, Coleman JR, Espie GS. Catalytic exchange of 18O from 13С18О-labelled CO2 by wild type cells and есаА, есаВ, and ссаА mutants оf the cyanobacteria Synechococcus PCC7942 and Synechocystis PCC6803. Can J Bot 1998; 76:1153–1160
     [Google Scholar]
  10. Wang D, Li Q, Li W, Xing J, Su Z. Improvement of succinate production by overexpression of a cyanobacterial carbonic anhydrase in Escherichia coli . Enzyme Microb Technol 2009; 45:491–497 [View Article]
     [Google Scholar]
  11. Rippka R, Stanier RY, Deruelles J, Herdman M, Waterbury JB. Generic assignments, strain histories and properties of pure cultures of cyanobacteria. Microbiology 1979; 111:1–61 [View Article]
     [Google Scholar]
  12. Mironov K, Los D. RNA Isolation from Synechocystis . Bio Protoc 2015; 5:e1428 [View Article]
     [Google Scholar]
  13. Green MR, Sambrook J. Molecular Cloning: A Laboratory Manual, 4th ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 2012
     [Google Scholar]
  14. Zimmerman SA, Tomb JF, Ferry JG. Characterization of CamH from Methanosarcina thermophila, founding member of a subclass of the γ class of carbonic anhydrases. J Bacteriol 2010; 192:1353–1360 [View Article][PubMed]
     [Google Scholar]
  15. McKinney MM, Parkinson A. A simple, non-chromatographic procedure to purify immunoglobulins from serum and ascites fluid. J Immunol Methods 1987; 96:271–278 [View Article][PubMed]
     [Google Scholar]
  16. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227:680–685 [View Article][PubMed]
     [Google Scholar]
  17. Wilbur KM, Anderson NG. Electrometric and colorimetric determination of carbonic anhydrase. J Biol Chem 1948; 176:147–154[PubMed]
     [Google Scholar]
  18. Kupriyanova EV, Cho SM, Park YI, Pronina NA, Los DA. The complete genome of a cyanobacterium from a soda lake reveals the presence of the components of CO2-concentrating mechanism. Photosynth Res 2016; 130:151–165 [View Article][PubMed]
     [Google Scholar]
  19. Pinto F, Pacheco CC, Ferreira D, Moradas-Ferreira P, Tamagnini P. Selection of suitable reference genes for RT-qPCR analyses in cyanobacteria. PLoS One 2012; 7:e34983 [View Article][PubMed]
     [Google Scholar]
  20. Thornton B, Basu C. Real-time PCR (qPCR) primer design using free online software. Biochem Mol Biol Educ 2011; 39:145–154 [View Article][PubMed]
     [Google Scholar]
  21. Marshall OJ. PerlPrimer: cross-platform, graphical primer design for standard, bisulphite and real-time PCR. Bioinformatics 2004; 20:2471–2472 [View Article][PubMed]
     [Google Scholar]
  22. von Heijne G. Signal sequences. The limits of variation. J Mol Biol 1985; 184:99–105[PubMed] [Crossref]
     [Google Scholar]
  23. Berks BC. The twin-arginine protein translocation pathway. Annu Rev Biochem 2015; 84:843–864 [View Article][PubMed]
     [Google Scholar]
  24. Okuyama T, Sato S, Zhu XL, Waheed A, Sly WS. Human carbonic anhydrase IV: cDNA cloning, sequence comparison, and expression in COS cell membranes. Proc Natl Acad Sci USA 1992; 89:1315–1319 [View Article][PubMed]
     [Google Scholar]
  25. Sly WS, Hu PY. Human carbonic anhydrases and carbonic anhydrase deficiencies. Annu Rev Biochem 1995; 64:375–401 [View Article][PubMed]
     [Google Scholar]
  26. Berg JM, Tymoczko JL, Stryer L. Biochemistry, 5th ed. NY: WH Freeman; 2002
     [Google Scholar]
  27. Denoncin K, Collet JF. Disulfide bond formation in the bacterial periplasm: major achievements and challenges ahead. Antioxid Redox Signal 2013; 19:63–71 [View Article][PubMed]
     [Google Scholar]
  28. Price GD, Badger MR. Expression of human carbonic anhydrase in the cyanobacterium Synechococcus PCC7942 creates a high CO2-requiring phenotype: evidence for a central role for carboxysomes in the CO2 concentrating mechanism. Plant Physiol 1989; 91:505–513 [View Article][PubMed]
     [Google Scholar]
  29. Schwarz D, Nodop A, Hüge J, Purfürst S, Forchhammer K et al. Metabolic and transcriptomic phenotyping of inorganic carbon acclimation in the cyanobacterium Synechococcus elongatus PCC 7942. Plant Physiol 2011; 155:1640–1655 [View Article][PubMed]
     [Google Scholar]
  30. Delepelaire P, Wandersman C. Protein export and secretion in Gram-negative bacteria. In Winkelmann G. (editor) Microbial Transport Systems Weinheim, Germany: Wiley-VCH; 2001 pp. 165–208 [Crossref]
     [Google Scholar]
  31. Park E, Rapoport TA. Mechanisms of Sec61/SecY-mediated protein translocation across membranes. Annu Rev Biophys 2012; 41:21–40 [View Article][PubMed]
     [Google Scholar]
  32. Stephenson K. Sec-dependent protein translocation across biological membranes: evolutionary conservation of an essential protein transport pathway (review). Mol Membr Biol 2005; 22:17–28 [View Article][PubMed]
     [Google Scholar]
  33. Payne SH, Bonissone S, Wu S, Brown RN, Ivankov DN et al. Unexpected diversity of signal peptides in prokaryotes. MBio 2012; 3:e00339-12 [View Article][PubMed]
     [Google Scholar]
  34. Kyte J, Doolittle RF. A simple method for displaying the hydropathic character of a protein. J Mol Biol 1982; 157:105–132 [View Article][PubMed]
     [Google Scholar]
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Putative extracellular α-class carbonic anhydrase, EcaA, of Synechococcus elongatus PCC 7942 is an active enzyme: a sequel to an old story
Microbiology 164, 576 (2018); https://doi.org/10.1099/mic.0.000634
/content/journal/micro/10.1099/mic.0.000634
/content/journal/micro/10.1099/mic.0.000634
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