The cyanide degrading nitrilase from Pseudomonas stutzeri AK61 is a two-fold symmetric, 14-subunit spiral.

The quaternary structure of the cyanide dihydratase from Pseudomonas stutzeri AK61 was determined by negative stain electron microscopy and three-dimensional reconstruction using the single particle technique. The structure is a spiral comprising 14 subunits with 2-fold symmetry. Interactions across the groove cause a decrease in the radius of the spiral at the ends and the resulting steric hindrance prevents the addition of further subunits. Similarity to two members of the nitrilase superfamily, the Nit domain of NitFhit and N-carbamyl-D-amino acid amidohydrolase, enabled the construction of a partial atomic model that could be unambiguously fitted to the stain envelope. The model suggests that interactions involving two significant insertions in the sequence relative to these structures leads to the left-handed spiral assembly.

[1]  G. Cohen.  Align : A program to superimpose protein coordinates, accounting for insertions and deletions , 1997 .

[2]  D. Matthews,et al.  Purification and characterization of cyanide hydratase from the phytopathogenic fungus Gloeocercospora sorghi. , 1992, Archives of biochemistry and biophysics.

[3]  I. Karube,et al.  Cloning and expression of a gene encoding cyanidase from Pseudomonas stutzeri AK61 , 1998, Applied Microbiology and Biotechnology.

[4]  R A Crowther,et al.  MRC image processing programs. , 1996, Journal of structural biology.

[5]  D T Jones,et al.  Protein secondary structure prediction based on position-specific scoring matrices. , 1999, Journal of molecular biology.

[6]  E A Merritt,et al.  Raster3D: photorealistic molecular graphics. , 1997, Methods in enzymology.

[7]  W. Fry,et al.  Hydrogen cyanide detoxification by Gloeocercospora sorghi , 1975 .

[8]  K. Yamamoto,et al.  Purification and characterization of nitrilase responsible for the enantioselective hydrolysis from Acinetobacter sp. AK 226. , 1991, Agricultural and biological chemistry.

[9]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[10]  D. Stalker,et al.  Purification and properties of a nitrilase specific for the herbicide bromoxynil and corresponding nucleotide sequence analysis of the bxn gene. , 1988, The Journal of biological chemistry.

[11]  Y. Ikenaka,et al.  Crystal structure of N-carbamyl-D-amino acid amidohydrolase with a novel catalytic framework common to amidohydrolases. , 2000, Structure.

[12]  Charlotte M. Deane,et al.  JOY: protein sequence-structure representation and analysis , 1998, Bioinform..

[13]  Laurent Joyeux,et al.  Efficiency of 2D alignment methods. , 2002, Ultramicroscopy.

[14]  David C. Jones,et al.  GenTHREADER: an efficient and reliable protein fold recognition method for genomic sequences. , 1999, Journal of molecular biology.

[15]  D. Cowan,et al.  Characterization of an inducible nitrilase from a thermophilic bacillus , 1999, Extremophiles.

[16]  Roland L. Dunbrack,et al.  Prediction of protein side-chain rotamers from a backbone-dependent rotamer library: a new homology modeling tool. , 1997, Journal of molecular biology.

[17]  S. Godtfredsen,et al.  Novel cyanide-hydrolyzing enzyme from Alcaligenes xylosoxidans subsp. denitrificans , 1991, Applied and environmental microbiology.

[18]  T. Bhalla,et al.  Asymmetric hydrolysis of α-aminonitriles to optically active amino acids by a nitrilase of Rhodococcus rhodochrous PA-34 , 1992, Applied Microbiology and Biotechnology.

[19]  Wolfgang Baumeister,et al.  A giant protease with a twist: the TPP II complex from Drosophila studied by electron microscopy , 2002, The EMBO journal.

[20]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[21]  D. Cowan,et al.  Biochemistry and biotechnology of mesophilic and thermophilic nitrile metabolizing enzymes , 1998, Extremophiles.

[22]  H. Yamada,et al.  Microbial transformations of nitriles , 1989 .

[23]  H. Yamada,et al.  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. , 1992, Biochemistry.

[24]  D. B. Harper.  Purification and properties of an unusual nitrilase from Nocardia N.C.I.B. 11216. , 1976, Biochemical Society transactions.

[25]  H. Yamada,et al.  Purification and characterization of a novel nitrilase of Rhodococcus rhodochrous K22 that acts on aliphatic nitriles , 1990, Journal of bacteriology.

[26]  D. B. Harper.  Microbial metabolism of aromatic nitriles. Enzymology of C-N cleavage by Nocardia sp. (Rhodochrous group) N.C.I.B. 11216. , 1977, The Biochemical journal.

[27]  A Leith,et al.  SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. , 1996, Journal of structural biology.

[28]  A. Mihoc,et al.  Mechanistic and structural studies on Rhodococcus ATCC 39484 nitrilase.“ , 1992, Biotechnology and applied biochemistry.

[29]  K. Gekko,et al.  Nitrilase of Rhodococcus rhodochrous J1. Conversion into the active form by subunit association. , 2000, European journal of biochemistry.

[30]  D. Rawlings,et al.  Isolation and characterization of a cyanide dihydratase from Bacillus pumilus C1 , 1993, Journal of bacteriology.

[31]  J Frank,et al.  Three-dimensional reconstruction of single particles embedded in ice. , 1992, Ultramicroscopy.

[32]  I. Karube,et al.  Cyanide hydrolysis in a cyanide-degrading bacterium, Pseudomonas stutzeri AK61, by cyanidase. , 1998, Microbiology.

[33]  A. Goldlust,et al.  Induction, purification, and characterization of the nitrilase of Fusarium oxysporum f. sp. melonis , 1989 .

[34]  E. Weiler,et al.  Molecular characterization of two cloned nitrilases from Arabidopsis thaliana: key enzymes in biosynthesis of the plant hormone indole-3-acetic acid. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[35]  C. Brenner.  Catalysis in the nitrilase superfamily. , 2002, Current opinion in structural biology.

[36]  Richard C. Willson,et al.  CynD, the Cyanide Dihydratase from Bacillus pumilus: Gene Cloning and Structural Studies , 2003, Applied and Environmental Microbiology.

[37]  C. Brenner,et al.  The nitrilase superfamily: classification, structure and function , 2001, Genome Biology.

[38]  M. Lawrence,et al.  CONSCRIPT: a program for generating electron density isosurfaces for presentation in protein crystallography , 2000 .

[39]  P. Chacón,et al.  Multi-resolution contour-based fitting of macromolecular structures. , 2002, Journal of molecular biology.

[40]  M. Heel,et al.  Exact filters for general geometry three dimensional reconstruction , 1986 .

[41]  J. Frank,et al.  A common-lines based method for determining orientations for N > 3 particle projections simultaneously. , 1996, Ultramicroscopy.

[42]  Y. Pekarsky,et al.  Crystal structure of the worm NitFhit Rosetta Stone protein reveals a Nit tetramer binding two Fhit dimers , 2000, Current Biology.

[43]  D. T. Brown,et al.  Purification and properties of cyanide hydratase from Fusarium lateritium and analysis of the corresponding chy1 gene. , 1993, Journal of general microbiology.