Catalytic mechanism of inulinase from Arthrobacter sp. S37 |
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Authors: | Kim Kyoung-Yun Nascimento Alessandro S Golubev Alexander M Polikarpov Igor Kim Chung-Sei Kang Su-Il Kim Su-Il |
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Institution: | a School of Agricultural Biotechnology, Seoul National University, San 56-1, Sinlim-Dong, Gwanak-Gu, Seoul 151-742, Republic of Korea b Instituto de Física, Universidade de São Paulo, Av. Trabalhador Sãocarlense, 400 CEP 13560-970, São Carlos, SP, Brazil c Petersburg Nuclear Physics Institute, Gatchina, St. Petersburg, 188300, Russia d International Environmental Research Center, Gwangju Institute of Science and Technology, 1 Oryong-Dong, Buk-Gu, Gwangju 500-712, Republic of Korea |
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Abstract: | Detailed catalytic roles of the conserved Glu323, Asp460, and Glu519 of Arthrobacter sp. S37 inulinase (EnIA), a member of the glycoside hydrolase family 32, were investigated by site-directed mutagenesis and pH-dependence studies of the enzyme efficiency and homology modeling were carried out for EnIA and for D460E mutant. The enzyme efficiency (kcat/Km) of the E323A and E519A mutants was significantly lower than that of the wild-type due to a substantial decrease in kcat, but not due to variations in Km, consistent with their putative roles as nucleophile and acid/base catalyst, respectively. The D460A mutant was totally inactive, whereas the D460E and D460N mutants were active to some extent, revealing Asp460 as a catalytic residue and demonstrating that the presence of a carboxylate group in this position is a prerequisite for catalysis. The pH-dependence studies indicated that the pKa of the acid/base catalyst decreased from 9.2 for the wild-type enzyme to 7.0 for the D460E mutant, implicating Asp460 as the residue that interacts with the acid/base catalyst Glu519 and elevates its pKa. Homology modeling and molecular dynamics simulation of the wild-type enzyme and the D460E mutant shed light on the structural roles of Glu323, Asp460, and Glu519 in the catalytic activity of the enzyme. |
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Keywords: | Glycoside hydrolase family 32 Endo-inulinase Arthrobacter sp S37 Catalytic mechanism Nucleophile Acid/base catalyst Homology modeling Molecular dynamics simulation |
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