ABC  Vol.3 No.3 , June 2013
Role of amino acid residues involved in the active cavity of proline iminopeptidase in catalytic activity
The proline iminopeptidase (PchPiPA) of the white-rot fungi Phanerochaete chrysosporium is an exopeptidase specific to catalyze hydrolysis of the N-terminal proline of peptides or proteins. Its catalytic cavity is comprised of a catalytic triad (Ser107, Asp264 and His292) and an oxyanion hole (His38, Gly39, Gly40 and Pro41). In this work, several amino acid residues involved in the catalytic cavity were selected for investigation of their influences on the catalytic activity by site-directed mutagenesis. It was shown that mutation of residues (Gly39 and Gly40) involved in oxyanion hole resulted in almost complete loss of catalytic activity largely due to changes in kcat. The other residues (Gly42 and Cys45) lined at the entrance of the active cavity also yielded a profound negative effect on the activity. Mutation of the other two residues Arg130 and Gly131 which were flanked spatially by the nucleophilic attacking active site of Ser107, caused different effects on the activity. R130Aincreased catalytic efficiency due to changes in both kcat and Km; while G131V decreased the value of kcat/Km mainly due to changes in kcat. And T111Aalso caused a negative effect on the kcat. Conclusively, these amino acid residues involved in active cavity were more susceptible to be negatively affected by mutation, suggested that the active cavity of proline iminopeptidase might evolve to be less plausible.

Cite this paper
Xing, K. and Feng, H. (2013) Role of amino acid residues involved in the active cavity of proline iminopeptidase in catalytic activity. Advances in Biological Chemistry, 3, 288-294. doi: 10.4236/abc.2013.33032.
[1]   Heikinheimo, P., Gold, A., Jeffries, C. and Ollis, D.L. (1999) Of barn owls and bankers: A lush variety of α/β hydrolases. Structure, 7, R141-R146. doi:10.1016/S0969-2126(99)80079-3

[2]   Sarid, S., Berger, A. and Katchalski, E. (1959) Proline iminopeptidase. Journal of Biological Chemistry, 234, 1740-1746.

[3]   Albertson, N.H. and Koomey, M. (1993) Molecular cloning and characterization of a proline iminopeptidase gene from Neisseria gonorrhoeae. Molecular Microbiology, 9, 1203-1211. doi:10.1111/j.1365-2958.1993.tb01249.x

[4]   Gilbert, C., Atlan, D., Banc, B. and Portalier, R. (1994) Proline iminopeptidase from Lactobacillus delbrueckii subsp. bulgaricus CNRZ 397: Purification and characterization. Microbiology, 140, 537-542. doi:10.1099/00221287-140-3-537

[5]   Kitazono, A., Kitano, A., Kabashima, T., Ito, K. and Yoshimoto, T. (1996) Prolyl aminopeptidase is also present in Enterobacteriaceae: Cloning and sequencing of the Hafnia alvei enzyme-gene and characterization of the expressed enzyme. Journal of Biochemistry, 119, 468-474. doi:10.1093/oxfordjournals.jbchem.a021265

[6]   Kitazono, A., Kitano, A., Tsuru, D. and Yoshimoto, T. (1994) Isolation and characterization of the prolyl aminopeptidase gene (pap) from Aeromonas sobria: comparison with the Bacillus coagulans enzyme. Journal of Biochemistry, 116, 818-825.

[7]   Li, N., Wu, J.-M., Zhang, L.-F., Zhang, Y.-Z. and Feng, H. (2010) Characterization of a unique proline iminopeptidase from white-rot basidiomycetes Phanerochaete chrysosporium. Biochimie, 92, 779-788. doi:10.1016/j.biochi.2010.02.022

[8]   Mahon, S., O’Donoghue, A.J., Goetz, D.H., Murray, P.G., Craik, C.S. and Tuohy, M.G. (2009) Characterization of a multimeric, eukaryotic prolyl aminopeptidase: An inducible and highly specific intracellular peptidase from the non-pathogenic fungus Talaromyces emersonii. Microbiology, 155, 3673-3682. doi:10.1099/mic.0.030940-0

[9]   Basten, D.E.J.W., Moers, A.P.H.A., van Ooyen, A.J.J. and Schaap, P.J. (2005) Characterisation of Aspergillus niger prolyl aminopeptidase. Molecular Genetics and Genomics, 272, 673-679. doi:10.1007/s00438-004-1094-5

[10]   Szawlowska, U., Grabowska, A., Zdunek-Zastocka, E. and Bielawski, W. (2012) TsPAP1 encodes a novel plant prolyl aminopeptidase whose expression is induced in response to suboptimal growth conditions. Biochemical and Biophysical Research Communications, 419, 104-109. doi:10.1016/j.bbrc.2012.01.140

[11]   Szaw1owska, U., Prus, W. and Bielawski, W. (2006) The molecular and biochemical characteristics of proline iminopeptidase from rye seedling (Secale cereal L.). Acta Physiologiae Plantarum, 28, 517-524.

[12]   Zhang, L., Jia, Y., Wang, L. and Wang, R. (2007) A proline iminopeptidase gene upregulated in plant by a LuxR homologue is essential for pathogenicity of Xanthomonas campestris pv. campestris. Molecular Microbiology, 65, 121-136. doi:10.1111/j.1365-2958.2007.05775.x

[13]   Allaker, R.P., Young, K.A. and Hardie, J.M. (1994) Rapid detection of proline iminopeptidase as an indicator of Eikenella corrodens. Letters of Applied Microbiology, 19, 325-327. doi:10.1111/j.1472-765X.1994.tb00466.x

[14]   Delauney, A.J. and Verma, D.P.S. (1993) Proline biosynthesis and osmoregulation in plant. The Plant Journal, 4, 215-223. doi:10.1046/j.1365-313X.1993.04020215.x

[15]   FitzGerald, R.J. and O’Cuinn, G., (2006) Enzymatic debittering of food protein hydrolysates. Biotechnology Advances. 24, 234-237. doi:10.1016/j.biotechadv.2005.11.002

[16]   Leenhouts, K., Bolhuis, A., Boot, J., Deutz, I., Toonen, M., Venema, J., Kok, J. and Ledeboer, A. (1998) Cloning, expression, and chromosomal stabilization of the Propionibacterium shermanii proline iminopeptidase gene (pip) for food-grade application in Lactococcus lactis. Applied and Environmental Microbiology, 64, 47364742.

[17]   Yamamoto, Y., Usuki, H., Kumagai, Y., Mukaihara, T., Yamosato, A. and Hatanaka, T. (2011) Synthesis of prolyl-hydroxyproline using prolyl aminopeptidase from Streptomyces aureofaciens TH-3. Process Biochemistry, 46, 1560-1564. doi:10.1016/j.procbio.2011.04.009

[18]   Yamomoto, Y., Usuki, H., Iwabuchi, M. and Hatanaka, T. (2010) Prolyl iminopeptidase from Streptomyces thermoluteus subsp. fuscus strain NBRC14270 and synthesis of proline-containing peptides. Applied Environmental Microbiology, 76, 6180-6185. doi:10.1128/AEM.01242-10

[19]   Rawlings, N.D., Barrett, A.J. and Bateman, A. (2010) MEROPS: The peptidase database. Nucleic Acids Research, 38, D227-D233. doi:10.1093/nar/gkp971

[20]   Medrano, F.J., Alonso, J., Garcia, J.L., Romero, A., Bode, W. and Gomis-Ru, F.X. (1998) Structure of proline iminopeptidase from Xanthomonas campestris pv. citri: A prototype for the prolyl oligopeptidase family. The EMBO Journal, 17, 1-9. doi:10.1093/emboj/17.1.1

[21]   Yoshimoto, T., Kabashim, T., Uchikawa, K., Unoue, T., Tanaka, N., Nakamura K.T., Tsuru, M. and Ito, K. (1999) Crystal structure of prolyl aminopeptidase from Serratia marcescens. Journal of Biochemistry, 126, 559-565. doi:10.1093/oxfordjournals.jbchem.a022486

[22]   Ito, K., Inoue, T., Kabashima, T., Kanada N., Huang, H.S., Ma, X., Azmi, N., Azab, E. and Yoshimoto, T. (2000) Substrate recognition mechanism of prolyl aminopeptidase from Serratia marcescens. Journal of Biochemistry, 128, 673-678. doi:10.1093/oxfordjournals.jbchem.a022800

[23]   Inoue, T., Ito, K., Tozaka, T., Hatakeyama, S., Tanaka, N., Nakamura, K.T. and Yoshimoto, T. (2003) Novel inhibitor for prolyl aminopeptidase from Serratia marcescens and studies on the mechanism of substrate recognition of the enzyme using the inhibitor. Archives of Biochemistry and Biophysics, 416, 147-154. doi:10.1016/S0003-9861(03)00293-5

[24]   Nakajima, Y., Ito, K., Sakata, M., Xu, Y., Nakashima, K., Matsubara, F., Katakeyama, S. and Yoshimoto, T. (2006) Unusual extra space at the active site and high activity for acetylated hydroxyproline of prolyl aminopeptidase from Serratia marcescens. Journal of Bacteriology, 188, 15991606. doi:10.1128/JB.188.4.1599-1606.2006

[25]   Kitazono, A., Ito, K. and Yoshimoto, T. (1994) Prolyl aminopeptidase is not a sulfhydryl enzyme: identification of the active serine residue by site-directed mutagenesis. Journal of Biochemistry, 116, 943945.

[26]   Morel, F., Gilbert, C., Geourjon, C., Frot-Coutaz, J., Portalier, R. and Atlan, D. (1999) The prolyl aminopeptidase from Lactobacillus delbrueckii subsp. Bulgaricus belongs to the α/β hydrolase fold family. Biochimia et Biophysica Acta, 1429, 501-505. doi:10.1016/S0167-4838(98)00264-7

[27]   Gall, M., Kourist, R., Schmidt, M., Bornscheuer and U.T. (2010) The role of the GGGX motif in determine the activity and enantioselectivity of pig liver esterase towards tertiary alcohols. Biocatalysis and Biotransformation, 28, 201-208. doi:10.3109/10242421003753803

[28]   Zhang, Y., Kua, J. and McCammon, J.A. (2002) Role of the catalytic triad and oxyanion hole in acetylcholinesterase catalysis: An ab initio QM/MM study. Journal of American Chemistry Society, 124, 10572-10577. doi:10.1021/ja020243m