Carboxyl proteinase from Pseudomonas defines a novel family of subtilisin-like enzymes

The crystal structure of a pepstatin-insensitive carboxyl proteinase from Pseudomonas sp. 101 (PSCP) has been solved by single-wavelength anomalous diffraction using the absorption peak of bromide anions. Structures of the uninhibited enzyme and of complexes with an inhibitor that was either covalently or noncovalently bound were refined at 1.0–1.4 Å resolution. The structure of PSCP comprises a single compact domain with a diameter of ∼55 Å, consisting of a seven-stranded parallel β-sheet flanked on both sides by a number of helices. The fold of PSCP is a superset of the subtilisin fold, and the covalently bound inhibitor is linked to the enzyme through a serine residue. Thus, the structure of PSCP defines a novel family of serine-carboxyl proteinases (defined as MEROPS S53) with a unique catalytic triad consisting of Glu 80, Asp 84 and Ser 287.

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

[2]  L. Delbaere,et al.  The 1.8 A structure of the complex between chymostatin and Streptomyces griseus protease A. A model for serine protease catalytic tetrahedral intermediates. , 1985, Journal of molecular biology.

[3]  Suzanne Fortier,et al.  Direct methods for solving macromolecular structures , 1998 .

[4]  Djordje Musil,et al.  The high-resolution X-ray crystal structure of the complex formed between subtilisin Carlsberg and eglin c, an elastase inhibitor from the leech Hirudo medicinalis Structural analysis, subtilisin structure and interface geometry , 1987 .

[5]  J. Kraut,et al.  Structure of Subtilisin BPN′ at 2.5 Å Resolution , 1969, Nature.

[6]  M. Ito,et al.  Identification of carboxyl residues in pepstatin-insensitive carboxyl proteinase from Pseudomonas sp. 101 that participate in catalysis and substrate binding. , 1999, Journal of biochemistry.

[7]  S. Murao,et al.  Purification and properties of a pepstatin-insensitive carboxyl proteinase from a gram-negative bacterium. , 1987, Biochimica et biophysica acta.

[8]  A Wlodawer,et al.  Practical experience with the use of halides for phasing macromolecular structures: a powerful tool for structural genomics. , 2001, Acta crystallographica. Section D, Biological crystallography.

[9]  S J Remington,et al.  Peptide aldehyde complexes with wheat serine carboxypeptidase II: implications for the catalytic mechanism and substrate specificity. , 1996, Journal of molecular biology.

[10]  G. Bricogne,et al.  [27] Maximum-likelihood heavy-atom parameter refinement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods. , 1997, Methods in enzymology.

[11]  K. Oda,et al.  Identification of Catalytic Residues of Pepstatin-insensitive Carboxyl Proteinases from Prokaryotes by Site-directed Mutagenesis* , 1999, The Journal of Biological Chemistry.

[12]  R. Huber,et al.  Accurate Bond and Angle Parameters for X-ray Protein Structure Refinement , 1991 .

[13]  J. Kraut,et al.  Subtilisin; a stereochemical mechanism involving transition-state stabilization. , 1972, Biochemistry.

[14]  M. Gribskov,et al.  A left‐handed crossover involved in amidohydrolase catalysis , 1993, FEBS letters.

[15]  Anastassis Perrakis,et al.  Automated protein model building combined with iterative structure refinement , 1999, Nature Structural Biology.

[16]  N. Rawlings,et al.  Tripeptidyl-peptidase I is apparently the CLN2 protein absent in classical late-infantile neuronal ceroid lipofuscinosis. , 1999, Biochimica et biophysica acta.

[17]  D. Davies,et al.  The structure and function of the aspartic proteinases. , 1990 .

[18]  P. Lobel,et al.  The Human CLN2 Protein/Tripeptidyl-Peptidase I Is a Serine Protease That Autoactivates at Acidic pH* , 2001, The Journal of Biological Chemistry.

[19]  T A Jones,et al.  Electron-density map interpretation. , 1997, Methods in enzymology.

[20]  P. Kuhn,et al.  The 0.78 A structure of a serine protease: Bacillus lentus subtilisin. , 1998, Biochemistry.

[21]  F. Young Biochemistry , 1955, The Indian Medical Gazette.

[22]  S. Takahashi,et al.  Cloning, nucleotide sequence, and expression of an isovaleryl pepstatin-insensitive carboxyl proteinase gene from Pseudomonas sp. 101. , 1994, The Journal of biological chemistry.

[23]  B. Dunn,et al.  Substrate specificity and kinetic properties of pepstatin-insensitive carboxyl proteinase from Pseudomonas sp. No. 101. , 1992, Biochimica et biophysica acta.

[24]  B. Dunn,et al.  Substrate specificity of pepstatin-insensitive carboxyl proteinase from Bacillus coagulans J-4. , 1998, Journal of biochemistry.

[25]  R. Donnelly,et al.  Association of mutations in a lysosomal protein with classical late-infantile neuronal ceroid lipofuscinosis. , 1997, Science.

[26]  T. Shin,et al.  Purification and characterization of kumamolysin, a novel thermostable pepstatin-insensitive carboxyl proteinase from Bacillus novosp. MN-32. , 1993, Journal of Biological Chemistry.

[27]  C. Sander,et al.  Protein structure comparison by alignment of distance matrices. , 1993, Journal of molecular biology.

[28]  G. Sheldrick,et al.  SHELXL: high-resolution refinement. , 1997, Methods in enzymology.

[29]  K D Cowtan,et al.  Phase combination and cross validation in iterated density-modification calculations. , 1996, Acta crystallographica. Section D, Biological crystallography.

[30]  T. Ahn,et al.  Pepstatin‐insensitive carboxyl proteinase: A biochemical marker for late lysosomes in Amoeba proteus , 1999 .

[31]  Mike Carson,et al.  RIBBONS 2.0 , 1991 .

[32]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[33]  A. Berger,et al.  On the size of the active site in proteases. I. Papain. , 1967, Biochemical and biophysical research communications.

[34]  A Wlodawer,et al.  Catalytic triads and their relatives. , 1998, Trends in biochemical sciences.