Possible role of inter-domain salt bridges in oligopeptidase B from Trypanosoma brucei: critical role of Glu172 of non-catalytic β-propeller domain in catalytic activity and Glu490 of catalytic domain in stability of OPB.
Oligopeptidase B (OPB) is a member of the prolyl oligopeptidase (POP) family of serine proteases. OPB in trypanosomes is an important virulence factor and potential pharmaceutical target. Characteristic structural features of POP family members include lack of a propeptide and presence of a β-propeller domain (PD), although the role of the β-PD has yet to be fully understood. In this work, residues Glu(172), Glu(490), Glu(524) and Arg(689) in Trypanosoma brucei OPB (Tb OPB), which are predicted to form inter-domain salt bridges, were substituted for Gln and Ala, respectively. These mutants were evaluated in terms of catalytic properties and stability. A negative effect on kcat/Km was obtained following mutation of Glu(172) or Arg(689). In contrast, the E490Q mutant exhibited markedly decreased thermal stability, although this mutation had less effect on catalytic properties compared to the E172Q and R689A mutants. Trypsin digestion showed that the boundary regions between the β-PD and catalytic domains (CDs) of the E490Q mutant are unfolded with heat treatment. These results indicated that Glu(490) in the CD plays a role in stabilization of Tb OPB, whereas Glu(172) in the β-PD is critical for the catalytic activity of Tb OPB.
Department of Biological Science and Technology, The University of Tokushima Graduate School, 2-1 Minamijosanjima, Tokushima 770-8506, Japan; Faculty of Science, Universiti Tunku Abdul Rahman, Jalam Universiti, Bandar Barat, 31900 Kampar, Perak D.R., Malaysia; and Department of Parasitology, Kurume University School of Medicine, 67 Asahi-machi, Kurume, Fukuoka 830-0011, Japan.
Junki
J
Fukumoto
Nor Ismaliza Mohd
NI
Ismail
Masaki
M
Kubo
Keita
K
Kinoshita
Masahiro
M
Inoue
Keizo
K
Yuasa
Makoto
M
Nishimoto
Hitoshi
H
Matsuki
Akihiko
A
Tsuji
D055162:Biocatalysis; D004795:Enzyme Stability; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D009154:Mutation; D017434:Protein Structure, Tertiary; D012492:Salts / Q000737:chemistry / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
D012492:Salts; D018698:Glutamic Acid; D012697:Serine Endopeptidases; C090703:oligopeptidase B
POP family; oligopeptidase B; salt bridge; thermal stability; β-propeller domain