Applied Microbiology and Biotechnology

, Volume 82, Issue 2, pp 271–278 | Cite as

The cyanide hydratase from Neurospora crassa forms a helix which has a dimeric repeat

  • Kyle C. Dent
  • Brandon W. Weber
  • Michael J. Benedik
  • B. Trevor Sewell
Biotechnologically Relevant Enzymes and Proteins


The fungal cyanide hydratases form a functionally specialized subset of the nitrilases which catalyze the hydrolysis of cyanide to formamide with high specificity. These hold great promise for the bioremediation of cyanide wastes. The low resolution (3.0 nm) three-dimensional reconstruction of negatively stained recombinant cyanide hydratase fibers from the saprophytic fungus Neurospora crassa by iterative helical real space reconstruction reveals that enzyme fibers display left-handed D1 S5.4 symmetry with a helical rise of 1.36 nm. This arrangement differs from previously characterized microbial nitrilases which demonstrate a structure built along similar principles but with a reduced helical twist. The cyanide hydratase assembly is stabilized by two dyadic interactions between dimers across the one-start helical groove. Docking of a homology-derived atomic model into the experimentally determined negative stain envelope suggests the location of charged residues which may form salt bridges and stabilize the helix.


Cyanide Nitrilase 3d protein reconstruction Cyanide hydratase 



We thank Professor Edward H. Egelman for his assistance with the IHRSR programs and interpretation of the helical power spectrum, Mohammed Jaffer for assistance with the electron microscope, Dr. Arvind Varsani for his assistance with expression, and the Carnegie Corporation of New York, the National Research Foundation, the University of Cape Town, the Texas Hazardous Waste Research Center, and the Robert A. Welch Foundation (A-1310) for financial support.


  1. Andrade J, Karmali A, Carrondo MA, Frazão C (2007) Structure of amidase from Pseudomonas aeruginosa showing a trapped acyl transfer reaction intermediate state. J Biol Chem 282:19598–19605CrossRefGoogle Scholar
  2. 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 CrossRefGoogle Scholar
  3. Baxter J, Cummings SP (2006) The current and future applications of microorganism in the bioremediation of cyanide contamination. Antonie Van Leeuwenhoek 90:1–17CrossRefGoogle Scholar
  4. Bower JM, Cohen FE, Dunbrack RL Jr (1997) Prediction of protein side-chain rotamers from a backbone-dependant rotamer library: a new homology modeling tool. J Mol Biol 267:1268–1282CrossRefGoogle Scholar
  5. Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  6. Brenner C (2002) Catalysis in the nitrilase superfamily. Curr Opin Struct Biol 12:775–782CrossRefGoogle Scholar
  7. Chacón P, Wriggers W (2002) Multi-resolution contour-based fitting of macromolecular structures. J Mol Biol 317:375–384CrossRefGoogle Scholar
  8. Cohen GH (1997) ALIGN: a program to superimpose protein coordinates, accounting for insertions and deletions. J Appl Crystall 30:1160–1161CrossRefGoogle Scholar
  9. Egelman EH (2000) A robust algorithm for the reconstruction of helical filaments using single-particle methods. Ultramicroscopy 85:225–234CrossRefGoogle Scholar
  10. Fiser A, Sali A (2003) MODELLER: generation and refinement of homology-based protein structure models. Methods Enzymol 374:463–493Google Scholar
  11. Fisher FB, Brown JS (1952) Colorimetric determination of cyanide in stack gas and waste water. Anal Chem 24:1440–1444CrossRefGoogle Scholar
  12. Frank J, Radermacher M, Penczek P, Zhu J, Li Y, Ladjadj M, Leith A (1996) SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J Struct Biol 116:190–199CrossRefGoogle Scholar
  13. Hung CL, Liu JH, Chiu WC, Huang SW, Hwang JK, Wang W-C (2007) Crystal structure of Helicobacter pylori formamidase AmiF reveals a cysteine–glutamate–lysine catalytic triad. J Biol Chem 282:12220–12229CrossRefGoogle Scholar
  14. Jandhyala D, Berman M, Meyers PR, Sewell BT, Willson RC, Benedik MJ (2003) Cyn D, the cyanide dihydratase from Bacillus pumillus: gene cloning and structural studies. Appl Environ Microbiol 69:4794–4805CrossRefGoogle Scholar
  15. Jandhyala DM, Wilson RC, Sewell BT, Benedik MJ (2005) Comparison of cyanide degrading nitrilases. Appl Microbiol Biotechnol 68:327–335CrossRefGoogle Scholar
  16. Jones DT (1999) GenTHREADER: an efficient and reliable protein fold recognition method for genomic sequences. J Mol Biol 287:797–815CrossRefGoogle Scholar
  17. Kimani SW, Agarkar VB, Cowan DA, Sayed MF-R, Sewell BT (2007) The crystal structure of an aliphatic amidase from Geobacillus pallidus RAPc8: evidence for a fourth nitrilase catalytic residue. Acta Crystallogr D63:1048–1058Google Scholar
  18. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefGoogle Scholar
  19. Ludtke SJ, Baldwin PR, Chiu W (1999) EMAN: semi-automated software for high-resolution single particle reconstructions. J Struct Biol 128:82–96CrossRefGoogle Scholar
  20. Lundgren S, Lohkamp B, Andersen B, Piskur J, Dobritzsch D (2008) The crystal structure of β-alanine synthase from Drosophila melanogaster reveals a homoactameric helical turn-like assembly. J Mol Biol 377:1544–1559CrossRefGoogle Scholar
  21. Makowski L, Caspar DLD (1980) The symmetries of filamentous phage particles. J Mol Biol 145:611–617Google Scholar
  22. McGuffin LJ, Jones DT (2003) Improvement of the GenTHREADER method for genomic fold recognition. Bioinformatics 19:874–881CrossRefGoogle Scholar
  23. Mueller P, Egorova K, Vorgias CE, Boutou E, Trauthwein H, Verseck S, Antranikian G (2006) Cloning, overexpression, and characterization of a thermoactive nitrilase from the hyperthermophilic archaeon Pyrococcus abyssi. Protein Expr Purif 47:672–681CrossRefGoogle Scholar
  24. 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 267:138–144CrossRefGoogle Scholar
  25. 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. Structure 8:729–737CrossRefGoogle Scholar
  26. O’Reilly C, Turner PD (2003) The nitrilases family of CN hydrolyzing enzymes—a comparative study. J Appl Microbiol 95:1161–1174CrossRefGoogle Scholar
  27. Pace HC, Brenner C (2001) The nitrilase superfamily: classification, structure and function. Genome Biol 2(1):REVIEWS0001CrossRefGoogle Scholar
  28. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612CrossRefGoogle Scholar
  29. Sakai N, Tajika Y, Yao M, Watanabe N, Tanaka I (2004) Crystal structure of hypothetical protein PH0642 from Pyrococcus Horikoshii at 1.6A resolution. Proteins Struct Funct Bioinform 57:869–873CrossRefGoogle Scholar
  30. Sali A, Blundell TL (1993) Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol 234:779–815CrossRefGoogle Scholar
  31. Sewell BT, Berman MN, Meyers PR, Jandhyala D, Benedik M (2003) The cyanide degrading nitrilase from Pseudomonas stutzeri AK61 is a two-fold symmetric, 14-subunit spiral. Structure 11:1–20CrossRefGoogle Scholar
  32. Sewell BT, Thuku RN, Zhang X, Benedik M (2005) The oligomeric structure of nitrilases: the effect of mutating interfacial residues on activity. Ann NY Acad Sci 1056:153–159CrossRefGoogle Scholar
  33. 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 15:283–302Google Scholar
  34. 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 274:2099–2018CrossRefGoogle Scholar
  35. Wang W, Hsu W, Chien F, Chen C (2001) Crystal structure and site-directed mutagenesis studies of N-Carbamoyl-D-amino-acid amidohydrolase from Agrobacterium radiobacter reveals a homotetramer and insight into a catalytic cleft. J Mol Biol 306:251–261CrossRefGoogle Scholar
  36. Wang YA, Yu X, Yip C, Strynadka NC, Egelman EH (2006) Structural polymorphism in bacterial EspA filaments revealed by Cryo-EM and an improved approach to helical reconstruction. Structure 14:1–8CrossRefGoogle Scholar
  37. Woodward JD, Weber BW, Scheffer MP, Benedik MJ, Hoenger A, Sewell BT (2008) Helical structure of unidirectionally shadowed fibres of cyanide hydratase from Gloeocercospora sorghi. J Struct Biol 161:111–119CrossRefGoogle Scholar
  38. Yang S, Yu X, Galkin VE, Egelman EH (2003) Issues of resolution and polymorphism in single-particle reconstruction. J Struct Biol 144:162–171CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Kyle C. Dent
    • 1
  • Brandon W. Weber
    • 2
  • Michael J. Benedik
    • 3
  • B. Trevor Sewell
    • 2
  1. 1.Department of Molecular and Cell BiologyUniversity of Cape TownRondeboschSouth Africa
  2. 2.Electron Microscope UnitUniversity of Cape TownRondeboschSouth Africa
  3. 3.Department of BiologyTexas A&M UniversityCollege StationUSA

Personalised recommendations