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-propel...

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Bibliographic Details
Published in:Journal of biochemistry (Tokyo) 2013-11, Vol.154 (5), p.465-473
Main Authors: Fukumoto, Junki, Ismail, Nor Ismaliza Mohd, Kubo, Masaki, Kinoshita, Keita, Inoue, Masahiro, Yuasa, Keizo, Nishimoto, Makoto, Matsuki, Hitoshi, Tsuji, Akihiko
Format: Article
Language:English
Subjects:
Biocatalysis
Enzyme Stability
Glutamic Acid - genetics
Glutamic Acid - metabolism
Hot Temperature
Mutation
Protein Structure, Tertiary
Salts - chemistry
Salts - metabolism
Serine Endopeptidases - chemistry
Serine Endopeptidases - metabolism
Trypanosoma brucei
Trypanosoma brucei brucei - enzymology
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Summary: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.
ISSN:0021-924X
1756-2651
DOI:10.1093/jb/mvt077