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Probing C-terminal interactions of the Pseudomonas stutzeri cyanide-degrading CynD protein


The cyanide dihydratases from Bacillus pumilus and Pseudomonas stutzeri share high amino acid sequence similarity throughout except for their highly divergent C-termini. However, deletion or exchange of the C-termini had different effects upon each enzyme. Here we extended previous studies and investigated how the C-terminus affects the activity and stability of three nitrilases, the cyanide dihydratases from B. pumilus (CynDpum) and P. stutzeri (CynDstut) and the cyanide hydratase from Neurospora crassa. Enzymes in which the C-terminal residues were deleted decreased in both activity and thermostability with increasing deletion lengths. However, CynDstut was more sensitive to such truncation than the other two enzymes. A domain of the P. stutzeri CynDstut C-terminus not found in the other enzymes, 306GERDST311, was shown to be necessary for functionality and explains the inactivity of the previously described CynDstut-pum hybrid. This suggests that the B. pumilus C-terminus, which lacks this motif, may have specific interactions elsewhere in the protein, preventing it from acting in trans on a heterologous CynD protein. We identify the dimerization interface A-surface region 195–206 (A2) from CynDpum as this interaction site. However, this A2 region did not rescue activity in C-terminally truncated CynDstutΔ302 or enhance the activity of full-length CynDstut and therefore does not act as a general stability motif.

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  1. Agarkar VB, Kimani SW, Cowan DA, Sayed MF, Sewell BT (2006) The quaternary structure of the amidase from Geobacillus pallidus RAPc8 is revealed by its crystal packing. Acta Crystallogr Sect F: Struct Biol Cryst Commun 62(Pt 12):1174–1178. doi:10.1107/S1744309106043855

    Article  CAS  Google Scholar 

  2. Andrade J, Karmali A, Carrondo MA, Frazao C (2007a) Crystallization, diffraction data collection and preliminary crystallographic analysis of hexagonal crystals of Pseudomonas aeruginosa amidase. Acta Crystallogr Sect F: Struct Biol Cryst Commun 63(Pt 3):214–216. doi:10.1107/S1744309107005830

    Article  CAS  Google Scholar 

  3. Andrade J, Karmali A, Carrondo MA, Frazao C (2007b) Structure of amidase from Pseudomonas aeruginosa showing a trapped acyl transfer reaction intermediate state. J Biol Chem 282(27):19598–19605. doi:10.1074/jbc.M701039200

    Article  CAS  PubMed  Google Scholar 

  4. Basile LJ, Willson RC, Sewell BT, Benedik MJ (2008) Genome mining of cyanide-degrading nitrilases from filamentous fungi. Appl Microbiol Biotechnol 80(3):427–435. doi:10.1007/s00253-008-1559-2

    Article  CAS  PubMed  Google Scholar 

  5. Dent KC, Weber BW, Benedik MJ, Sewell BT (2009) The cyanide hydratase from Neurospora crassa forms a helix which has a dimeric repeat. Appl Microbiol Biotechnol 82(2):271–278. doi:10.1007/s00253-008-1735-4

    Article  CAS  PubMed  Google Scholar 

  6. Hung CL, Liu JH, Chiu WC, Huang SW, Hwang JK, Wang WC (2007) Crystal structure of Helicobacter pylori formamidase AmiF reveals a cysteine–glutamate–lysine catalytic triad. J Biol Chem 282(16):12220–12229. doi:10.1074/jbc.M609134200

  7. Jandhyala D, Berman M, Meyers PR, Sewell BT, Willson RC, Benedik MJ (2003) CynD, the cyanide dihydratase from Bacillus pumilus: gene cloning and structural studies. Appl Environ Microbiol 69(8):4794–4805

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  8. Jandhyala DM, Willson RC, Sewell BT, Benedik MJ (2005) Comparison of cyanide-degrading nitrilases. Appl Microbiol Biotechnol 68(3):327–335. doi:10.1007/s00253-005-1903-8

    Article  CAS  PubMed  Google Scholar 

  9. Kimani SW, Agarkar VB, Cowan DA, Sayed MF, Sewell BT (2007) Structure of an aliphatic amidase from Geobacillus pallidus RAPc8. Acta Crystallogr D Biol Crystallogr 63(Pt 10):1048–1058. doi:10.1107/S090744490703836X

    Article  CAS  PubMed  Google Scholar 

  10. Kiziak C, Klein J, Stolz A (2007) Influence of different carboxy-terminal mutations on the substrate-, reaction- and enantiospecificity of the arylacetonitrilase from Pseudomonas fluorescens EBC191. Protein Eng Des Sel 20(8):385–396. doi:10.1093/Protein/Gzm032

    Article  CAS  PubMed  Google Scholar 

  11. Kumaran D, Eswaramoorthy S, Gerchman SE, Kycia H, Studier FW, Swaminathan S (2003) Crystal structure of a putative CN hydrolase from yeast. Proteins Struct Funct Genet 52(2):283–291. doi:10.1002/Prot.10417

    Article  CAS  PubMed  Google Scholar 

  12. Lundgren S, Lohkamp B, Andersen B, Piskur J, Dobritzsch D (2008) The crystal structure of beta-alanine synthase from Drosophila melanogaster reveals a homooctameric helical turn-like assembly. J Mol Biol 377(5):1544–1559. doi:10.1016/j.jmb.2008.02.011

    Article  CAS  PubMed  Google Scholar 

  13. Meyers PR, Rawlings DE, Woods DR, Lindsey GG (1993) Isolation and characterization of a cyanide dihydratase from Bacillus pumilus C1. J Bacteriol 175(19):6105–6112

    PubMed Central  CAS  PubMed  Google Scholar 

  14. Nagasawa T, Wieser M, Nakamura T, Iwahara H, Yoshida T, Gekko K (2000) Nitrilase of Rhodococcus rhodochrous J1. Conversion into the active form by subunit association. Eur J Biochem 267(1):138–144

    Article  CAS  PubMed  Google Scholar 

  15. Nakai T, Hasegawa T, Yamashita E, Yamamoto M, Kumasaka T, Ueki T, Nanba H, Ikenaka Y, Takahashi S, Sato M, Tsukihara T (2000) Crystal structure of N-carbamyl-d-amino acid amidohydrolase with a novel catalytic framework common to amidohydrolases. Struct Fold Des 8(7):729–737. doi:10.1016/S0969-2126(00)00160-X

    Article  CAS  Google Scholar 

  16. Nichols M, Willits C (1934) Reactions of Nessler's solution. J Am Chem Soc 56:769–774

    Article  CAS  Google Scholar 

  17. Pace HC, Brenner C (2001) The nitrilase superfamily: classification, structure and function. Genome Biol 2(1):REVIEWS0001

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  18. Pace HC, Hodawadekar SC, Draganescu A, Huang J, Bieganowski P, Pekarsky Y, Croce CM, Brenner C (2000) Crystal structure of the worm NitFhit Rosetta Stone protein reveals a Nit tetramer binding two Fhit dimers. Curr Biol 10(15):907–917. doi:10.1016/S0960-9822(00)00621-7

    Article  CAS  PubMed  Google Scholar 

  19. Park J (2014) Regions involved in the oligomerization and activity of the spiral forming nitrilase Cyanide Dihydratase. Ph.D. Dissertation, Texas A&M University, USA

  20. Rinagelova A, Kaplan O, Vesela AB, Chmatal M, Krenkova A, Plihal O, Pasquarelli F, Cantarella M, Martinkova L (2014) Cyanide hydratase from Aspergillus niger K10: overproduction in Escherichia coli, purification, characterization and use in continuous cyanide degradation. Process Biochem 49(3):445–450. doi:10.1016/J.Procbio.2013.12.008

    Article  CAS  Google Scholar 

  21. Sewell BT, Berman MN, Meyers PR, Jandhyala D, Benedik MJ (2003) The cyanide degrading nitrilase from Pseudomonas stutzeri AK61 is a two-fold symmetric, 14-subunit spiral. Structure 11(11):1413–1422

    Article  CAS  PubMed  Google Scholar 

  22. Sewell BT, Thuku RN, Zhang X, Benedik MJ (2005) Oligomeric structure of nitrilases: effect of mutating interfacial residues on activity. Ann N Y Acad Sci 1056:153–159

    Article  CAS  PubMed  Google Scholar 

  23. Stevenson DE, Feng R, Dumas F, Groleau D, Mihoc A, Storer AC (1992) Mechanistic and structural studies on Rhodococcus ATCC 39484 nitrilase. Biotechnol Appl Biochem 15(3):283–302

    CAS  PubMed  Google Scholar 

  24. Thimann KV, Mahadevan S (1964) Nitrilase. I. Occurrence preparation + general properties of enzyme. Arch Biochem Biophys 105(1):133. doi:10.1016/0003-9861(64)90244-9

    Article  CAS  PubMed  Google Scholar 

  25. Thuku RN, Brady D, Benedik MJ, Sewell BT (2009) Microbial nitrilases: versatile, spiral forming, industrial enzymes. J Appl Microbiol 106(3):703–727. doi:10.1111/j.1365-2672.2008.03941.x

    Article  CAS  PubMed  Google Scholar 

  26. Thuku RN, Weber BW, Varsani A, Sewell BT (2007) Post-translational cleavage of recombinantly expressed nitrilase from Rhodococcus rhodochrous J1 yields a stable, active helical form. FEBS J 274(8):2099–2108. doi:10.1111/j.1742-4658.2007.05752.x

    Article  CAS  PubMed  Google Scholar 

  27. Wang L, Watermeyer JM, Mulelu AE, Sewell BT, Benedik MJ (2012) Engineering pH-tolerant mutants of a cyanide dihydratase. Appl Microbiol Biotechnol 94(1):131–140. doi:10.1007/s00253-011-3620-9

    Article  CAS  PubMed  Google Scholar 

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The financial supports of The Welch Foundation (A1310) and the Texas Hazardous Waste Research Center are gratefully acknowledged.

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The authors declare that they have no conflict of interest.

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Correspondence to Michael J. Benedik.

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Crum, M.AN., Park, J.M., Mulelu, A.E. et al. Probing C-terminal interactions of the Pseudomonas stutzeri cyanide-degrading CynD protein. Appl Microbiol Biotechnol 99, 3093–3102 (2015).

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  • Cyanide dihydratase
  • Nitrilase
  • Cyanide
  • Bioremediation