Novel inhibitor for prolyl aminopeptidase from Serratia marcescens and studies on the mechanism of substrate recognition of the enzyme using the inhibitor.

Prolyl aminopeptidase from Serratia marcescens hydrolyzed x-beta-naphthylamides (x=prolyl, alanyl, sarcosinyl, L-alpha-aminobutylyl, and norvalyl), which suggested that the enzyme has a pocket for a five-member ring. Based on the substrate specificity, novel inhibitors of Pro, Ala, and Sar having 2-tert-butyl-[1,3,4]oxadiazole (TBODA) were synthesized. The K(i) value of Pro-TBODA, Ala-TBODA, and Sar-TBODA was 0.5 microM, 1.6 microM, and 12mM, respectively. The crystal structure of enzyme-Pro-TBODA complex was determined. Pro-TBODA was located at the active site. Four electrostatic interactions were located between the enzyme and the amino group of Pro inhibitors (Glu204:0E1-N:Inh, Glu204:0E2-N:Inh, Glu232:0E1-N:Inh, and Gly46:O-N:Inh), and the residue of the inhibitors was inserted into the hydrophobic pocket composed of Phe139, Leu141, Leu146, Tyr149, Tyr150, and Phe236. The roles of Phe139, Tyr149, and Phe236 in the hydrophobic pocket and Glu204 and Glu232 in the electrostatic interactions were confirmed by site-directed mutagenesis, which indicated that the molecular recognition of proline is achieved through four electrostatic interactions and an insertion in the hydrophobic pocket of the enzyme.

[1]  T. Yoshimoto,et al.  Prolyl aminopeptidase is also present in Enterobacteriaceae: cloning and sequencing of the Hafnia alvei enzyme-gene and characterization of the expressed enzyme. , 1996, Journal of biochemistry.

[2]  The Mechanism of Substrate Recognition of Pyroglutamyl-peptidase I from Bacillus amyloliquefaciens as Determined by X-ray Crystallography and Site-directed Mutagenesis* , 2001, The Journal of Biological Chemistry.

[3]  T. Yoshimoto,et al.  Crystal structure of prolyl aminopeptidase from Serratia marcescens. , 1999, Journal of biochemistry.

[4]  T. Yoshimoto,et al.  Prolyl aminopeptidase gene from Flavobacterium meningosepticum: cloning, purification of the expressed enzyme, and analysis of its sequence. , 1996, Archives of biochemistry and biophysics.

[5]  E A Merritt,et al.  Raster3D Version 2.0. A program for photorealistic molecular graphics. , 1994, Acta crystallographica. Section D, Biological crystallography.

[6]  M. Koomey,et al.  Molecular cloning and characterization of a proline iminopeptidase gene from Neisseria gonorrhoeae , 1993, Molecular microbiology.

[7]  D E McRee,et al.  XtalView/Xfit--A versatile program for manipulating atomic coordinates and electron density. , 1999, Journal of structural biology.

[8]  T. Yoshimoto,et al.  Prolyl aminopeptidase is not a sulfhydryl enzyme: identification of the active serine residue by site-directed mutagenesis. , 1994, Journal of biochemistry.

[9]  T. Yoshimoto,et al.  Cloning, sequencing, and high expression of the proline iminopeptidase gene from Bacillus coagulans , 1992, Journal of bacteriology.

[10]  L. W. Spruce,et al.  Development of orally active nonpeptidic inhibitors of human neutrophil elastase. , 2001, Journal of medicinal chemistry.

[11]  T Takeuchi,et al.  Diprotins A and B, inhibitors of dipeptidyl aminopeptidase IV, produced by bacteria. , 1984, The Journal of antibiotics.

[12]  T. Katayama,et al.  The Initiator Function of DnaA Protein Is Negatively Regulated by the Sliding Clamp of the E. coli Chromosomal Replicase , 1998, Cell.

[13]  F. S. Mathews,et al.  Monomeric Sarcosine Oxidase: 1. Flavin Reactivity and Active Site Binding Determinants†,‡ , 2000 .

[14]  R. Mentlein,et al.  Prolyl aminopeptidase from rat brain and kidney. Action on peptides and identification as leucyl aminopeptidase. , 1990, European journal of biochemistry.

[15]  T. Yoshimoto,et al.  Prolyl aminopeptidase from Serratia marcescens: cloning of the enzyme gene and crystallization of the expressed enzyme. , 1997, Journal of biochemistry.

[16]  J. Alonso,et al.  Proline iminopeptidase gene from Xanthomonas campestris pv. citri. , 1996, Microbiology.

[17]  Sven Branner,et al.  Crystal structure of human dipeptidyl peptidase IV/CD26 in complex with a substrate analog , 2003, Nature Structural Biology.

[18]  D. Atlan,et al.  Cloning, sequencing and characterization of the pepIP gene encoding a proline iminopeptidase from Lactobacillus delbrueckii subsp. bulgaricus CNRZ 397. , 1994, Microbiology.

[19]  T. Yoshimoto,et al.  Isolation and characterization of the prolyl aminopeptidase gene (pap) from Aeromonas sobria: comparison with the Bacillus coagulans enzyme. , 1994, Journal of Biochemistry (Tokyo).

[20]  T. Yoshimoto,et al.  Substrate recognition mechanism of prolyl aminopeptidase from Serratia marcescens. , 2000, Journal of biochemistry.

[21]  T. Yoshimoto,et al.  Post-proline cleaving enzyme and post-proline dipeptidyl aminopeptidase. Comparison of two peptidases with high specificity for proline residues. , 1978, The Journal of biological chemistry.

[22]  M. Harada,et al.  Purification and properties of dipeptidyl peptidase II from rat kidney. , 1983, Biochimica et biophysica acta.

[23]  T. Yoshimoto,et al.  Postproline cleaving enzyme: identification as serine protease using active site specific inhibitors. , 1977, Biochemistry.

[24]  T. Yoshimoto,et al.  Thiazolidine derivatives as potent inhibitors specific for prolyl endopeptidase. , 1988, Journal of biochemistry.

[25]  Charles A. Kennedy,et al.  Studies on Proline Boronic Acid Dipeptide Inhibitors of Dipeptidyl Peptidase IV: Identification of a Cyclic Species Containing a B-N Bond , 1994 .

[26]  W. Bachovchin,et al.  Inhibition of IgA1 proteinases from Neisseria gonorrhoeae and Hemophilus influenzae by peptide prolyl boronic acids. , 1990, The Journal of biological chemistry.

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

[28]  W. Baumeister,et al.  The proteasome from Thermoplasma acidophilum is neither a cysteine nor a serine protease , 1995, FEBS letters.

[29]  László Polgár,et al.  Prolyl Oligopeptidase An Unusual β-Propeller Domain Regulates Proteolysis , 1998, Cell.