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Journal of Bacteriology, December 2008, p. 7819-7829, Vol. 190, No. 23
0021-9193/08/$08.00+0     doi:10.1128/JB.02010-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Dipeptidyl Aminopeptidase IV from Stenotrophomonas maltophilia Exhibits Activity against a Substrate Containing a 4-Hydroxyproline Residue{triangledown}

Yoshitaka Nakajima,1 Kiyoshi Ito,1* Tsubasa Toshima,1 Takashi Egawa,1 Heng Zheng,1,{dagger} Hiroshi Oyama,1,{ddagger} Yu-Fan Wu,1 Eiji Takahashi,2 Kiyoshi Kyono,2 and Tadashi Yoshimoto1

Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-Machi, Nagasaki 852-8521, Japan,1 Tanabe Seiyaku Co., Ltd., 16-89 Kashima, 3-Chome, Yodogawa-Ku, Osaka 532-8505, Japan2

Received 27 December 2007/ Accepted 10 September 2008

The crystal structure of dipeptidyl aminopeptidase IV from Stenotrophomonas maltophilia was determined at 2.8-Å resolution by the multiple isomorphous replacement method, using platinum and selenomethionine derivatives. The crystals belong to space group P43212, with unit cell parameters a = b = 105.9 Å and c = 161.9 Å. Dipeptidyl aminopeptidase IV is a homodimer, and the subunit structure is composed of two domains, namely, N-terminal β-propeller and C-terminal catalytic domains. At the active site, a hydrophobic pocket to accommodate a proline residue of the substrate is conserved as well as those of mammalian enzymes. Stenotrophomonas dipeptidyl aminopeptidase IV exhibited activity toward a substrate containing a 4-hydroxyproline residue at the second position from the N terminus. In the Stenotrophomonas enzyme, one of the residues composing the hydrophobic pocket at the active site is changed to Asn611 from the corresponding residue of Tyr631 in the porcine enzyme, which showed very low activity against the substrate containing 4-hydroxyproline. The N611Y mutant enzyme was generated by site-directed mutagenesis. The activity of this mutant enzyme toward a substrate containing 4-hydroxyproline decreased to 30.6% of that of the wild-type enzyme. Accordingly, it was considered that Asn611 would be one of the major factors involved in the recognition of substrates containing 4-hydroxyproline.

* Corresponding author. Mailing address: Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan. Phone: 81-95-819-2436. Fax: 81-95-819-2478. E-mail: k-ito{at}nagasaki-u.ac.jp

{triangledown} Published ahead of print on 26 September 2008.

{dagger} Present address: School of Life Science and Technology, China Pharmaceutical University, 24 Tongjia Street, Nanjing 210009, People's Republic of China.

{ddagger} Present address: Department of Immunobiology, Nihon Pharmaceutical University, 10281 Komuro, Ina-cho, Kitaadachi-gun, Saitama 362-0806, Japan.

Journal of Bacteriology, December 2008, p. 7819-7829, Vol. 190, No. 23
0021-9193/08/$08.00+0     doi:10.1128/JB.02010-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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