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Journal of Biochemistry Advance Access originally published online on October 6, 2008
Journal of Biochemistry 2008 144(6):771-780; doi:10.1093/jb/mvn130
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© The Authors 2008. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

Saturation Mutagenesis reveals that GLU54 of Norovirus 3C-like Protease is not Essential for the Proteolytic Activity

Yuichi Someya*, Naokazu Takeda and Takaji Wakita

Department of Virology II, National Institute of Infectious Diseases, Musashi-Murayama, Tokyo 208-0011, Japan

*To whom correspondence should be addressed. Tel: +81-42-561-0771, Fax: +81-42-561-4729, E-mail: someya{at}nih.go.jp

Received May 28, 2008; Accepted September 22, 2008


  Abstract

The norovirus 3C-like protease is a member of the chymotrypsin-like serine protease superfamily. Previous characterization of its crystal structure has implicated the Glu54–His30–Cys139 triad in the catalysis. In the present study, the Glu54 residue of the protease was subjected to site-saturation mutagenesis, with the result that nearly half of the mutants retained the significant proteolytic activity. It was suggested that a carboxylate at position 54 was not essential for the activity. The in vitro assays of the proteolysis revealed that most of Glu54 mutants retained relatively high proteolytic activity. When the Glu54 mutation was combined with the Ser mutation of the Cys139 residue, a nucleophile, only the Asp54 and Gln54 mutations showed proteolytic activity comparable to that of the Ser139 single mutant, suggesting that a hydrogen bond between Glu54 and His30 was critical in the Ser139 background. These results suggested that the mechanism of the proteolysis by the wild-type norovirus 3C-like protease was different from that of typical chymotrypsin-like serine proteases.

Key Words: 3C-like protease, catalytic triad, norovirus, serine-like cysteine protease, serine protease

Abbreviations: DNP, 2,4-dinitrophenyl; GST, glutathione S-transferase; NMA, 2-(N-methyl amino)benzoyl; NTPase, nucleotide triphosphatase; VPg, genome-linked viral protein


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Y. Someya and N. Takeda
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[Abstract] [Full Text] [PDF]


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