Various nitrilase structure projects

Various nitrilase structure projects

Introduction

The study of the function and structure of nitrilases is in its infancy. Most work to date has been done by biotechnologists who have sought to exploit the versatile lys-cys-glu catalytic triad for the manufacture of acids of industrial importance from nitriles or for the detoxification of cyanide. Our contribution to date has been the determination of the quaternary structure of two cyanide dihydratases, from Bacillus pumilus and from Pseudomonas stutzeri by a combination of 3D EM, homology modelling and fitting. The structures have a novel, defined length, spiral form comprising 18 and 14 subunits respectively. We have proposed a model for the termination of the spiral based on our structure and have made predictions about the existence and nature of a new inter subunit contact which gives rise to the spiral.

The structural work we have done is not of sufficiently high resolution to visualise the interface or indeed the active site at atomic resolution - but it does give insight into the reasons for the persistent failure to crystallize these enzymes and suggests strategies to overcome this bottleneck. One goal of our future work is to address these aspects, however our structure also suggests that the nitrilase may be the core of a larger multienzyme complex and pursuing this line might give insight into the biological role of these fascinating enzymes.

What we know about nitrilases

1. Structure

Knowledge at the atomic level is based on the crystal structures of some distantly related superfamily members. Our own papers describe the interesting quaternary structure of these enzymes.

2. Occurrence

By far the majority of papers simply note the occurrence of nitrilases in different organisms.

3. Sequences

It is not easy to find all the nitrilase superfamily sequences in the database. A comprehensive search was compiled by Pace and Brenner which is slightly out of date.

Summary of proposed projects

Four different projects are proposed:

1. The structural effects of mutations on nitrilase homologues

The model proposed by Sewell et al (2003) makes certain specific predictions about the location and role of various parts of these enzymes with respect to the formation of spiral structures. It is proposed that a series of mutations be made to test the model. It is envisaged that this approach will give insight into the details of the structure of the spiral and the interaction between the quaternary structure and activity of these enzymes. Furthermore, it is hoped that one of the smaller complexes will crystallize allowing the visualization of the active site and the predicted, spiral forming "C" surface.

2. Details of the structural transition in Bacillus pumilus cyanide dihydratase

This nitrilase forms defined length (16-18 sbunit) spirals in the pH range 6-8. However at pH 5.4 it forms long helical rods. It is proposed that the structural change is driven by the interaction of a charged histidine group with a carboxyl group. The goal of the project is the identification of the charged groups involved and the characterization of the molecular re-arrangements that accompany the gross structural changes.

3. Structure and identification of large multi-enzyme complexes involving nitrilases

4. The structure of the nitrilase from Rhodococcus rhodochrous J1.

Nagasawa et al (2000) have found that isolated dimers of the related nitrilase from Rhodococcus rhodochrous J1 are inactive. However in the presence of certain substrates they assemble to form an active decamer. ( A decamer is required to produce one turn of the spiral.) This behaviour is commonly observed in nitrilases from Rhodococcus sp. and is highly suggestive that the formation of the quaternary structure alters the active site. Crystals of J1 nitrilase in the absence of substrate have previously been obtained. The goal of this project would be to obtain an atomic structure in the absence of substrate and a 3DEM reconstruction of the oligomer in the presence of substrate.

1. The structural effects of mutations on nitrilase homologues.

We have suggested an alignment of three cyanide degrading nitrilases to the crystallographically determined structures of Nit and DCase which was primarily created using GenTHREADER.

We have also aligned the structures of Nit and DCase using ALIGN. (see superpose.pdb)

From this we have concluded that insertions and deletions in the cyanide degrading enzymes occur in externally located loops and that there are two major insertions, one major deletion and a substantial C-terminal extension.

An important difference between our enzymes, and indeed the majority of nitrilases, and the solved structures of superfamily members is the formation of spiral oligomers comprising specific numbers of subunits - but different numbers of subunits (ranging from 10-18) in different enzymes. We have postulated that the interaction that leads to the formation of the oligomer is due to an 15 amino acid insertion found in the loop between the beta strands labelled NS9 and NS10 in Nit. This, we postulate, leads to the formation of an interface (which we have called the C surface) which has pseudo two-fold symmetry. Indeed based on our model the insertion between NS2 and NH2 may also contribute to the interface and the deletion between NS5b and NS6a is essential as it would result in a clash preventing the formation of the C surface.

Our next step is to try and visualize the postulated C surface and to establish whether our postulates are correct. We have made (or intend making) a series of mutants in both the P. stutzeri and B. pumilus enzymes that:

  • disrupt the A surface - the point being to make dimers which preserve the C surface and crystallize.
  • remove the 15 amino acid insertion which we beleive is responsible for the C surface and verify that "Nit-like" dimers are formed
  • truncate the C-terminal extension so that its structural or other purpose can be determined.

To date the following mutants have been made and are ready for expression from pET expression vectors.

Enzyme

mutation

activity

# oligomers

P. stutzeri Y285stop No ?
P. stutzeri Q296stop No ?
P. stutzeri R303stop No ?
P. stutzeri K304stop Yes ?
P. stutzeri S310stop No ?
P. stutzeri Y241D + C244D No ?
P. stutzeri Del M260-F274 No ?
B. pumilus Y245stop No ?
B. pumilus M293stop Yes ?
B. pumilus K303stop Yes ?
G. sorghi Y217D ? ?
G. sorghi Y217E ? ?

The task of the student will be to express and purify the proteins, assess their oligomerization status by column chromatography and/or EM, set up crystallizations of any dimers or tetramers and solve the structure of one of the successful crystallizations. Much of the work will involve collaboration with Professor Michael Benedik at the University of Houston. Professor Benedik will be visiting in January 2004.

3. Identification and structure of large multi-enzyme complexes involving nitrilases

or "What protein is associated with the cyanide degrading nitrilase from Bacillus pumilus?"

Cyanide degrading nitrilases occur in a number of prokaryotic and eukaryotic microorganisms. Their biological role is generally unknown. They have attracted interest as agents for bioremediation of cyanide spills. There is also substantial interest in the use of closely related enzymes in biological catalysts. There is indirect evidence that the nitrilase plays a role in sporulation in the case of B. pumilus.

We have studied the structure of the nitrilase and have discovered that it has an 18 subunit spiral arrangement. This is the first report of this type of quaternary structure which has the effect of concentrating the nitrilase in a specific location and of creating a scaffold to which other proteins can attach.

The aim of this project is to find whether another protein is complexed with the nitrilase and if so to find out as much as possible about this protein.

There are a number of leads that point in the direction of there being such a protein:

  • We have seen vestiges of density at specific locations on EM reconstructions of nitrilase prepared from the native organism (but not on the recombinant protein). This indicates low occupancy of attached proteins.
  • In C. elegans and Drosophila the nitrilase exists as a fusion protein.
  • In C. elegans the nitrilase is a tetramer associating via a beta sheet. This beta sheet interface is exposed on the outside of the quaternary helix in our model and is this free to associate with other proteins. The location of the beta sheet corresponds with the observed vestigial density.
  • In the case of Bacillus sp. strain OxB-1 the gene for phenylacetaldoxime dehydratase (which cleaves an aldoxime to make a nitrile) is in the same gene cluster as the nitrilase which is a close homologue of ours. This genetic relationship is not the case in B. pumilus but there is the suggestion that two enzymes which could form part of a pathway are in close association.

Our initial plan is to try and pull intact complexes out of the native organism using immuno-affinity chromatography. But a number of other strategies are possible including affinity chromatography using immobilised purified recombinant enzyme.

The initial plan calls for:

  1. Attachment of chicken antibodies prepared against recombinant B. pumilus to a CL Sepharose matrix and using this as the basis of an affinity column.
  2. Preparation of a culture of B. pumilus, homogenization of this culture and passing it over the column.
  3. Analysis of high affinity entities to see if any contain the nitrilase and hopefully another protein.
  4. Visualization of the attached entity in the electron microscope.
  5. Characterization of the other protein by, SDS electrophoresis, MALDI, trypsin cleavage + MALDI and database searching.
  6. Analysis of the details of binding using BIACORE.

There is a great deal that may go wrong with this plan but there are a variety of alternative sub projects including, for example, using different species of nitrilase containing bacteria, using different affinity matrices, using electron microscopy to debug the various stages. And there are alternative routes depending on the specific interests of the student - for example - three dimensional reconstruction of the complex rather than the BIACORE experiments.

We have prepared the following resources for a student selecting this project:

  • Overexpression vectors for three different nitrilases (both with and without his tags).
  • Antibodies from chickens
  • Eggs from which further antibodies may be extracted.
  • Two species of bacteria (B. pumilus and Pseudomonas stutzeri)

Techniques

Antibody characterization, preparation and use of immunoaffinity columns, SDS gel electrophoresis, electron microscopy, preparation of bacterial cultures, preparation of protein from overexpression systems, MALDI, working with enzymes, using bioinformatics databases, BIACORE etc.

Literature

  1. Pace, H.C., Hodawadekar, S.C., Draganescu, A., Huang, J., Bieganowski, P., Pekarsky, Y., Croce, C.M. and Brenner, C.: Crystal Structure of the Worm NitFhit Rosetta Stone Protein Reveals a Nit Tetramer Binding Two Fhit Dimers. Current Biology, 10: 607-617, 2000.
  2. H.C. Pace & C. Brenner: The Nitrilase Superfamily: Classification, Structure and Function. Genome Biology, 2: reviews 0001.1-0001.9, 2001
  3. C. Brenner, "Catalysis in the Nitrilase Superfamily," Current Opinion in Structural Biology, 6: 775-782, 2002.

References

  1. Almatawah QA, Cramp R and Cowan DA (1999) Characterization of an inducible nitrilase from a thermophilic bacillus. Extremophiles 3, 283-291.
  2. Bandyopadathy AK, Nagasawa T, Asano Y, Fujishiro K, Tani Y and Yamada H (1986) Purification and characterization of benzonitrilases from Arthrobacter sp. strain J-1. Applied and Environmental Microbiology 51, 302-306.
  3. Banerjee A, Sharma R and Banerjee UC (2002) The nitrile-degrading enzymes: current status and future prospects. Applied Microbiology and Biotechnology 60, 33-44.
  4. Barclay M, Tett VA and Knowles CJ (1998) Metabolism and enzymology of cyanide/metallocyanide biodegradation by Fusarium solani under neutral and acidic conditions. Enzyme and Microbial Technology 23, 321-330.
  5. Bartling D, Seedorf M, Schmidt RC and Weiler EW (1994) Molecular characterization of two cloned nitrilases from Arabidopsis thaliana: key enzymes in biosynthesis of the plant hormone indole-3-acetic acid. Proceedings of the National Academy of Sciences U S A 91, 6021-5.
  6. Bengis-Garber C and Gutman AL (1989) Selective hydrolysis of dinitriles into cyano-carboxylic acids by Rhodococcus rhodochrous NCIB 11216. Applied Microbiology and Biotechnology 32, 11-16.
  7. Bhalla T, Miura A, Wakamoto A, Ohba Y and Furuhashi K (1992) Asymmetric hydrolysis of á-aminonitrilase to optically active amino acids by a nitrilase of Rhodococcus rhodochrous PA-34. Applied Microbiology and Biotechnology 37, 184-190.
  8. Bollag DM and Edelstein SJ (1991) Protein Methods. Wiley-Liss, Inc., New York, 96-115.
  9. Bradford, M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry. 72, 248-254.
  10. Brenner C (2002) Catalysis in the nitrilase superfamily. Current Opinion in Structural Biology 12, 775-782.
  11. Brown DT, Turner PD and O'Reilly C (1995) Expression of the cyanide hydratase enzyme from Fusarium lateritium in Escherichia coli and identification of an essential cysteine residue. FEMS Microbiology Letters 134, 143-146.
  12. Chen CY, Chiu WC, Liu JS, Hsu WH and Wang WC (2003) Structural basis for catalysis and substrate specificity of Agrobacterium radiobacter N-carbamoyl-D-amino-acid amidohydrolase. Journal of Biological Chemistry, epub ahead of print.
  13. Cluness MJ, Turner PD, Clements E, Brown DT and O'Reilly C (1993) Purification and properties of cyanide hydratase from Fusarium lateritium and analysis of the corresponding chy1 gene. Journal of General Microbiology 139, 1807-1815.
  14. Crowther, R. A., Henderson, R. and Smith, J. M. (1996) MRC image processing programs. Journal of Structural Biology 116, 9-16.
  15. Dadd MR, Claridge TD, Walton R, Pettman AJ and Knowles CJ (2001) Regioselective biotransformation of the dinitrile compounds 2-, 3- and 4- (cyanomethyl) benzonitrile by the soil bacterium Rhodococcus rhodochrous LL100-21. Enzyme and Microbial Technology 29, 20-27.
  16. Davies GE and Stark GR (1970) Use of dimethyl Suberimidate, a Cross-Linking Reagent, in Studying the Subunit Structure of Oligomeric Proteins. Proceedings of the National Academy of Sciences U S A. 66, 651-656.
  17. Dubey SK and Holmes DS (1995) Biological cyanide destruction mediated by microorganisms. World Journal of Microbiology and Biotechnology 11, 257- 265.
  18. Effenberger F and Osswald S (2001) Selective hydrolysis of aliphatic dinitriles to monocarboxylic acids by a nitrilase from Arabidopsis thaliana. Synthesis 1866- 1872.
  19. Egelman E (2000) A robust algorithm for the reconstruction of helical filaments using single particle methods. Ultramicroscopy 85, 225-234.
  20. Farnaud S, Tata R, Sohi MK, Wan T, Brown PR and Sutton BJ (1999) Evidence that cysteine-166 is the active-site nucleophile of Pseudomonas aeruginosa amidase: crystallization and preliminary X-ray diffraction analysis of the enzyme. Biochemical Journal 340, 711-714.
  21. Fisher FB and Brown JS (1952) Colorimetric determination of cyanide in stack gas and waste water. Analytical Chemistry 24, 1440-1444.
  22. Frank J, Radermacher M, Penczek P, Zhu J, Li Y, Ladjadj M and Leith A (1996a) SPIDER and WEB: Processing and Visualization of Images in 3D Electron Microscopy and Related Fields. Journal of Structural Biology 116, 190-199.
  23. Frank J (1996b) Three-dimensional Electron Microscopy of Macromolecular Assemblies.Academic Press, San Diego.
  24. Fry WE and Munch DC (1975). Hydrogen cyanide detoxification by Gloeocercospora sorghi. Physiological Plant Pathology 7, 23-33.
  25. Goldlust A and Bohak Z (1989) Induction, purification, and characterization of the nitrilase of Fusarium oxysporum f. sp. melonis. Biotechnology and Applied Biochemistry 11, 581-601.
  26. Gradley ML and Knowles CJ (1994) Asymmetric hydrolysis of chiral nitriles by Rhodococcus rhodochrous NCIMB 11216 nitrilase. Biotechnology Letters 16, 41-46.
  27. Harper DB (1977a) Microbial metabolism of aromatic nitriles. Enzymology of C- N cleavage by Nocardia sp. (Rhodococcus group) N.C.I.B. 11216. Biochemical Journal 165, 309-319.
  28. Harper DB (1977b) Fungal degradation of aromatic nitriles. Enzymology of C-N cleavage by Fusarium solani. Biochemical Journal 167, 685-92.
  29. Harper DB (1985) Characterization of a nitrilase from Nocardia sp. (Rhodochrous group) N.C.I.B. 11215, using p-hydroxybenzonitrile as sole carbon source. International Journal of Biochemistry, 17, 677-83.
  30. Harauz G and van Heel M (1986) Exact filters for general geometry three dimensional reconstruction. Optik 73, 146-156.
  31. Hook RH and Robinson WG (1964) Ricinine nitrilase II. Purification and properties. Journal of Biological Chemistry 239, 4263-4267 (see also Robinson and Hook, 1964).
  32. Hoyle AJ, Bunch AW, and Knowles CJ (1998) The nitrilases of Rhodococcus rhodochrous NCIMB 11216. Enzyme and Microbial Technology 23, 475-482.
  33. Ingvorsen K, Højer-Pedersen B and Godtfredsen SE (1991) Novel cyanide hydrolysing enzyme from Alcaligenes xylosoxidans subsp. denitrificans. Applied and Environmental Microbiology 57, 1783-1789.
  34. Jandhyala DM (2002) Cyanide degrading nitrilases for detoxification of cyanide containing waste waters. PhD thesis, University of Houston, 77-78.
  35. Jandhyala D, Berman MN, Meyers PR, Sewell BT, Willson RC and Benedik MJ (2003) CynD, the cyanide dihydratase from Bacillus pumilus: Gene cloning and structural studies. Applied and Environmental Microbiology, 69, 4794-4805.
  36. Jones DT (1999) GenTHREADER: an efficient and reliable protein fold recognition method for genomic sequences. Journal of Molecular Biology 287, 797-815.
  37. Joyeux L and Penczek PA (2002) Efficiency of 2D alignment methods. Ultramicroscopy 92, 33-46.
  38. Kobayashi M, Nagasawa T and Yamada H (1988) Regiospecific hydrolysis of dinitrile compounds by nitrilase from Rhodococcus rhodochrous J1. Applied Microbiology and Biotechnology 29, 231-233.
  39. Kobayashi M, Nagasawa T and Yamada H (1989) Nitrilase of Rhodococcus rhodochrous J1. Purification and characterization. European Journal of Biochemistry 182, 349-56.
  40. Kobayashi M, Yanaka N, Nagasawa T and Yamada H (1990a) Purification and characterization of a novel nitrilase of Rhodococcus rhodochrous K22 that acts on aliphatic nitriles. Journal of Bacteriology 172, 4807-15.
  41. Kobayashi M, Yanaka N, Nagasawa T and Yamada H (1990b) Monohydrolysis of an aliphatic dinitrile compound by nitrilase from Rhodococcus rhodochrous K22. Tetrahedron 46, 5587-5590.
  42. Kobayashi M, Komeda H, Yanaka N, Nagasawa T and Yamada H (1992a) Nitrilase from Rhodococcus rhodochrous J1. Sequencing and overexpression of the gene and identification of an essential cysteine residue. Journal of Biological Chemistry. 267, 20746-51.
  43. Kobayashi M, Yanaka N, Nagasawa T and Yamada H (1992b) Primary structure of an aliphatic nitrile-degrading enzyme, aliphatic nitrilase, from Rhodococcus rhodochrous K22 and expression of its gene and identification of its active site residue. Biochemistry 31, 9000-9007.
  44. Kobayashi M, Izui H, Nagasawa T and Yamada H (1993) Nitrilase in biosynthesis of the plant hormone indole-3-acetic acid from indole-3- acetonitrile: cloning of the Alcaligenes gene and site-directed mutagenesis of cysteine residues. Proceedings of the National Academy of Sciences U S A 90, 247-251.
  45. Kobayashi M and Shimizu S (1994) Versatile nitrilases: Nitrile-hydrolysing enzymes. FEMS Microbiology letters 120, 217-224.
  46. Kobayashi M, Goda M and Shimizu S (1998) Nitrilase catalyzes amide hydrolysis as well as nitrile hydrolysis. Biochemical and Biophysical Research communications 253, 662-666.
  47. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.
  48. Layh N, Parratt J and Willetts A (1998) Characterization and partial purification of an enantioselective arylacetonitrilase from Pseudomonas fluorescens DSM 7155. Journal of Molecular Catalysis B: Enzymatic 5, 467-474.
  49. Lévy-Schil S, Soubrier F, Crutz-Le Coq AM, Faucher D, Crouzet J and Pétré D (1995) Aliphatic nitrilase from a soil-isolated Comamonas testosteroni sp.: gene cloning and overexpression, purification and primary structure. Gene 161, 15-20.
  50. Mahadevan S and Thimann KV (1964) Nitrilase II. Substrate specificity and possible mode of action. Archives of Biochemistry and Biophysics 107, 62-68.
  51. Meyers PR, Gokool P, Rawlings DE and Woods DR (1991) An efficient cyanide-degrading Bacillus pumilus strain. Journal of General Microbiology 137, 1397-1400.
  52. Meyers P, Rawlings, DE, Woods, DR and Lindsey, GG (1993a) Isolation and characterization of a cyanide dihydratase from Bacillus pumilus C1. Journal of Bacteriology 175, 6105-6112.
  53. Meyers PR (1993b) Cyanide degradation by Bacillus pumilus C1: Cellular and Molecular Characterization. PhD thesis, University of Cape Town.
  54. Mitchell CG, Anderson SC and el-Mansi EM (1995) Purification and characterization of citrate synthase isoenzymes from Pseudomonas aeruginosa. Biochemical Journal 309, 507-511.
  55. Nakai T, Hasgawa T, Yamashita E, Yamamoto M, Kamasuka T, Ueki T, Nanba H, Ikenaka Y, Takahashi S, Sato M and Tsukihara T (2000) Crystal structure of N-carbamyl-D-amino acid amidohydrolase with a novel catalytic framework common to amidohydrolases. Structure 8, 729-737.
  56. Nagasawa T, Mauger J and Yamada H (1990) A novel nitrilase, arylacetonitrilase, of Alcaligenes faecalis JM3. Purification and characterization. European Journal of Biochemistry. 194, 765-72.
  57. Nagasawa T and Yamada H (1995) Microbial production of Commodity chemicals. Pure and Applied Chemistry 67, 1241-1256.
  58. Nagasawa T, Wieser M, Nakamura T, Iwahara H, Yoshida T and Gekko K (2000) Nitrilase of Rhodococcus rhodochrous J1 - Conversion into the active form by subunit association. European Journal of Biochemistry 267, 138-144.
  59. Nolan L, Harnedy P, Hearne A and O'Reilly C (2003) The cyanide hydratase enzyme of Fusarium lateritium also has nitrilase activity. FEMS Microbiology Letters, 221, 161-165.
  60. Novo C, Farnaud S, Tata R, Clemente A and Brown PR (2002) Support for a three-dimensional structure predicting a Cys-Glu-Lys catalytic triad for Pseudomonas aeruginosa amidase comes from site-directed mutagenesis and mutations altering substrate specificity. Journal of Biochemistry 365, 731-738.
  61. Osswald S, Wajant H and Effenberger F (2002) Characterization and synthetic applications of recombinant AtNIT1 from Arabidopsis thaliana. European Journal of Biochemistry 269, 680-687.
  62. Pace HC, Hodawadekar SC, Draganescu A, Huang J, Bieganowski P, Pekarsky Y, Croce CM and Brenner C (2000) Crystal structure of the worm NitFhit Rosetta Stone protein reveals a Nit tetramer binding two Fhit dimers. Current Biology, 10, 907-917.
  63. Pace HC and Brenner C (2001) The nitrilase superfamily: classification, structure and function. Genome Biology 2, reviews0001.1-0001.9
  64. Penczek P, Rademacher M, and Frank J (1992) Three-dimensional reconstruction of single particles embedded in ice. Ultramicroscopy 40, 33-53.
  65. Penczek P, Zhu J and Frank J (1996) A common-lines based method for determining orientations for N>3 particle projections simultaneously. Ultramicroscopy, 63, 205-218.
  66. Piotrowski M, Schonfelder S and Weiler EW (2001) The Arabidopsis thaliana isozyme NIT4 and its orthologs in tobacco encode beta-cyano-L-alanine hydratase/nitrilase. Journal of Biological Chemistry 276, 2616-2621.
  67. Price B, Chang P, Jandhyala DM, Benedik M and Sewell BT (2002) The quaternary structure of Gloeocercospora sorghi nitrilase (cyanide hydratase) as revealed by negative staining. Proceedings of the 15th International Congress on Electron Microscopy, 565-566.
  68. Richards FM (1974) The interpretation of protein structures: total volume, group volume distributions and packing density Journal of Molecular Biology 82, 1-14.
  69. Robinson WG and Hook RH (1964) Ricinine nitrilase I. Reaction product and substrate specificity. Journal of Biological Chemistry 239, 4257-4262.
  70. Rockel B, Peters J, Kuhlmorgen B, Glaeser RM and Baumeister W (2002) A giant protease with a twist: the TPP II complex from Drosophila studied by electron microscopy. EMBO Journal 21, 5979-5984.
  71. Romão MJ, Turk D, Gomis-Rüth F-X and Huber R (1992) Crystal structure analysis, refinement and enzymatic reaction mechanism of N- carbamoylsarcosine amidohydrolase from Arthrobacter sp. at 2.0 Å resolution. Journal of Molecular Biology, 226, 1111-1130.
  72. Stalker DM and McBride KE (1987) Cloning and Expression in Escherichia coli of a Klebsiella ozaenae plasmid-borne gene encoding a nitrilase specific for the herbicide Bromoxynil. Journal of Bacteriology 169, 955-960.
  73. Stalker DM, Malyj LD and McBride KE (1988a) Purification and properties of a nitrilase specific for the herbicide bromoxynil and corresponding nucleotide sequence analysis of the bxn gene. Journal of Biological Chemistry 263, 6310-4.
  74. Stalker DM, McBride KE and Malyj LD (1988b) Herbicide resistance in transgenic plants expressing a bacterial detoxification gene. Science 242, 419-423.
  75. Stevenson DE, Feng R and Storer AC (1990) Detection of covalent enzyme- substrate complexes of nitrilase by ion-spray mass spectroscopy. FEBS Letters 277, 112-114.
  76. Stevenson DE, Feng R, Dumas F, Groleau D, Mihoc A and Storer AC (1992) Mechanistic and structural studies on Rhodococcus ATCC 39484 nitrilase. Biotechnology and Applied Biochemistry. 15, 283-302.
  77. Thimann KV and Mahadevan S (1964) Nitrilase I. Occurrence, preparation, and general properties of the enyzme. Archives of Biochemistry and Biophysics 105, 133-141 (see also Mahadevan and Thimann (1964)).
  78. Tong EK and Duckworth HW (1975) The quaternary structure of citrate synthase
    from Escherichia coli K12. Biochemistry 14, 235-241.
  79. Wang P, Matthews DE and VanEtten HD (1992a) Purification and characterization of cyanide hydratase from the phytopathogenic fungus Gloeocercospora sorghi. Archives of Biochemistry and Biophysics 298, 569-575.
  80. Wang P and VanEtten HD (1992b) Cloning and properties of a cyanide hydratase gene from the phytopathogenic fungus Gloeocercospora sorghi. Biochemical and Biophysical Research Communications 187, 1048-1054.
  81. Wang W-C, Hsu W-H, Chien F-T and Chen C-Y (2001) Crystal structure and site-directed mutagensis studies of N-Carbamoyl-D-amino-acid amidohydrolase from Agrobacterium radiobacter reveals a homotetramer and insight into a catalytic cleft. Journal of Molecular Biology 306, 251-261.
  82. Wang P, Sandock RW and vanEtten HD (1999) Disruption of the cyanide hydratase gene in Gloeocercospora sorghi increases its sensitivity to the phytoanticipin cyanide but does not affect its pathogenicity on the cyanogenic plant sorghum. Fungal Genetics and Biology 28, 126-134.
  83. Watanabe A, Yano K, Ikebukuro K and Karube I (1998a) Cyanide hydrolysis in a cyanide-degrading bacterium, Pseudomonas stutzeri AK61, by cyanidase. Microbiology 144, 1677-1682.
  84. Watanabe A, Yano K, Ikebukuro K and Karube I (1998b) Cloning and expression of a gene encoding cyanidase from Pseudomonas stutzeri AK61 Applied Microbiology and Biotechnology 50, 93-97.
  85. Watanabe A, Yano K, Ikebukuro K and Karube I (1998c) Investigation of the potential active site of a cyanide dihydratase using site-directed mutagenesis. Biochimica et Biophysica Acta 1382, 1-4.
  86. Watanabe Y, Iwakura M, Tokushige M and Eguchi G (1981) Studies on Aspartase. VII. Subunit arrangement of Escherichia coli Aspartase Biochimica et Biophysica Acta, 661, 261-266.
  87. Yamamoto K and Komatsu K (1991) Purification and characterization of nitrilase responsible for enantioselective hydrolysis from Acinetobacter sp. AK 226. Agricultural and Biological Chemistry 55, 1459-1466.