56NISR Research GRANT
2005 Research Grant

Study on Proline-specific Aminopeptidases and Their Application on Food Processing
Kiyoshi ITO
Graduate School of Biomedical Sciences, Nagasaki University

Research aims
Hydrolyzed vegetable proteins produced through hydrochloric acid hydrolysis (acid-HVP) are widely used as
the savoury ingredient and have been added to many food.
Recently, it became a serious problem because acid-HVP is
contaminated with a group of chemicals known as chloropropanols which include 1, 3-dichloropropanol (1,3-DCP)and 3-monochloropropane-1, 2-diol (3-MCPD). Thesecompounds are by-products in acid-HVP and have insalutary effects on human body. During the acid hydrolysis,components of fats and oils in the starting materials may bechlorinated at high temperature to form chloropropanols. Incontrast, enzymatic hydrolysis of proteins produces no suchcompounds and therefore it is considered safe. Proteases
have been utilized for food processing for long time andthere are a large number of basic researches on the enzymatic hydrolysis of proteins or peptides into specific peptides. However, the enzymatic method cannot digest proteins completely into amino acids and has not yet been applied as a practical method. This is due to the fact that ordinary peptidases cannot or hardly act on peptide bonds
around proline residues. Our main purpose here is to understand the substrate recognition and hydrolysis mechanismof the proline-specific aminopeptidases in order to developa practical enzymatic method to achieve a complete digestion of proteins.
Methods
Prolyl aminopeptidase (PAP) from Serratia marcescens,prolyl tripeptidyl aminopeptidase (PTP) from Porphyromonas gingivalis, and aminopeptidase N (APN), which
can release a N-terminal proline residue of the substrate

peptide in Escherichia coli, were used as proline-specificaminopeptidases. Wild-type forms of PAP1) and APN2)
were expressed in E. coli. N-terminal truncated form ofPTP (PTPD 39) lacking the membrane-anchoring region
was expressed as a soluble form of enzyme3). Enzymes
were purified to homogeneity and crystallized by using ahanging drop vapor diffusion method. The enzyme structures were determined by X-ray crystallography using datacollected at synchrotron facilities4–6).
Results(1) Prolyl aminopeptidase: The presence of a hydrophobic pocket that recognizes the N-terminal proline residue
was clarified from the crystal structures of the PAP-inhibitor complexes. Interestingly, there was an unusual extra
space at the bottom of the hydrophobic pocket where proline residue is fixed in the PAP. Moreover, the 4-acetyloxylgroup of the substrate can be accommodated in this space.
(2) Prolyl tripeptidyl aminopeptidase: PTP was a dimmer
and the each monomer is consisted of b -propellar and catalytic domains, and a large cavity between the domains.
Those structural features are common in the prolyloligopeptidase (POP) family. The catalytic triad was conserved in the catalytic domain and a proline-recognizinghydrophobic pocket was identified. The PTP structure issimilar to dipeptidyl aminopeptidase IV (DPIV), which recognizes a proline residue at the penultimate position fromthe N-terminus and releases a dipeptide. However, there
were characteristic differences which were related to thesubstrate specificity of tripeptidyl activity. The N-terminal
amino group was recognized by two carboxyl groups of
Glu205 and Glu206 from a helix in dipeptidyl aminopeptidase IV. In prolyl tripeptidyl aminopeptidase, however, the57

Glu205 (located in the loop) and Glu636 were found tocarry out this function. The loop structure provides sufficient space to accommodate three N-terminal residues(Xaa-Xaa-Pro) of substrates.
(3) Aminopeptidase N: APN was composed of four domains: an N-terminal b -domain, a catalytc domain, a middle b -domain, and a C-terminal a -domain. The structure ofthe catalytic domain exhibits similarity to thermolysin. The
groove on the catalytic domain that contains the active siteis covered by the C-terminal a -domain, and a large cavity
is formed inside the protein. However, there existed a small
hole at the center of the C-terminal a -domain. The amino
terminus of bestatin was recognized by Glu121 andGlu264. Glu298 and Tyr381 are considered to be involvedin peptide cleavage. A difference revealed between the ligand-free form and the enzyme-bestatin complex indicatedthat Met260 functions as a cushion to accept substrates
with different amino terminal sizes, resulting in the broad
substrate specificity of this enzyme.
Conclusion
We have clarified the crystal structures of two prolinespecific aminopeptidases and of the aminopeptidase N
which shows a broad substrate specificity including a pro-line residue. Based on these structures, proline recognition
mechanism can be discussed from the view of three dimensional structure. These findings may be able to overcome
the difficulty that ordinary peptidases can not or hardly act
on peptide bonds around proline residues and will afford insights into a base in developing a practical and efficient enzymatic method to digest proteins completely into aminoacids.
References1) Inoue, T., Ito, K., Tosaka, T., Hatakeyama S., Tanaka, N., Nakamura,K. T., and Yoshimoto, T. (2003) Arch. Biochem. Biophys. 416:147–154
2) Onohara, Y., Nakajima, Y., Ito, K., Xu, Y., Nakashima, K., Ito, T.,and Yoshimoto, T. (2006) Acta Cryst. F62: 699–701
3) Nakajima, Y., Ito, K., Xu, Y., Yamada, N., Onohara, Y., Ito, T., andYoshimoto, T. (2005) Acta Cryst. F61: 1046–1048
4) Nakajima, Y., Ito, K., Sakata, M., Xu, Y., Nakashima, K., Matsubara,F., Hatakeyama, S., and Yoshimoto, T. (2006) J. Bacteriol., 188:1599–1606
5) Ito, K., Nakajima, Y., Xu, Y., Yamada, N., Onohara, Y., Ito, T., Matsubara, F., Kabashima, T., Nakayama, K., and Yoshimoto, T. (2006)
J. Mol. Biol., 362: 228–240
6) Ito, K., Nakajima, Y., Onohara, Y., Takeo, M., Nakashima, K., Matsubara, F., Ito, T., and Yoshimoto, T. (2006) J. Biol. Chem., 281:33664–33676