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Journal of Biochemistry Advance Access published online on October 9, 2009
Journal of Biochemistry, doi:10.1093/jb/mvp156
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© The authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

A Critical Role for highly Conserved GLU610 Residue of Oligopeptidase B from Trypanosoma Brucei in Thermal Stability

Nor Ismaliza Mohd Ismail1, Tsuyoshi Yuasa1, Keizo Yuasa1, Yuko Nambu1,3, Makoto Nisimoto1, Masaki Goto1, Hitoshi Matsuki1, Masahiro Inoue2, Masami Nagahama1,4 and Akihiko Tsuji1,*

1Department of Biological Science and Technology, University of Tokushima Graduate School, 2-1 Minamijosanjima, Tokushima 770-8506, Japan
2Department of Parasitology, Kurume University School of Medicine, 67 Asahi-machi, Kurume, Fukuoka 830-0011, Japan
3Present address: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Gokasyo, Uji, Kyoto, 611-0011, Japan
4Present address: Department of Molecular Biochemistry, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan

*Address correspondence to: Dr. Akihiko Tsuji, 1Department of Biological Science and Technology, University of Tokushima Graduate School, 2-1 Minamijosanjima, Tokushima 770-8506, Japan Tel:+81-88-656-7526; Fax:+81-88-655-3861; E-mail: tsuji{at}bio.tokushima-u.ac.jp

Received July 17, 2009; Accepted September 26, 2009


  Abstract

Oligopeptidase B from Trypanosoma brucei (Tb OPB) is a virulence factor and therapeutic target in African sleeping sickness. Three glutamic acid residues at positions 607, 609 and 610 of the catalytic domain are highly conserved in the OPB subfamily. In this study, the roles of Glu607, Glu609 and Glu610 in Tb OPB were investigated by site-directed mutagenesis. A striking effect on kcat/Km was obtained following mutation of Glu607 to glutamine. In contrast, the heat stability of Tb OPB decreased markedly following the single mutation of Glu610 to glutamine, although this mutation had significantly less effect on catalytic properties compared with the Glu607 mutation. Although no differences were found in the tertiary and secondary structures between wild-type OPB and the E610Q mutant prior to heat treatment, the E610Q mutant is inactivated more rapidly than wild-type OPB following heat treatment in a manner correlating with its attendant structural changes. Trypsin digestion showed that the boundary regions between the β-propeller and catalytic domain of the E610Q mutant are unfolded with heat treatment. It is concluded that Glu607 is essential for the catalytic activity of Tb OPB and that Glu610 plays a critical role in stabilization rather than catalytic activity despite their close proximity.

Key Words: Prolyl oligopeptidase, Oligopeptidase B, propellar domain, Thermal stability, Trypanosoma


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