Site-directed mutagenesis of possible catalytic residues of cellobiose 2-epimerase from Ruminococcus albus
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概要
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Cellobiose 2-epimerase (EC 5.1.3.11) from Ruminococcus albus (RaCE) catalyzes the reversible epimerization of cellobiose and lactose to 4-O-β-D-glucopyranosyl-D-mannose and 4-O-β-D-galactopyranosyl-D-mannose (epilactose). Based on the structure-based amino acid sequence alignment with N-acetyl-D-glucosamine 2-epimerases (EC 5.1.3.8) from porcine kidney and Anabaena sp. CH1 and on the computer-aided model building of the tertiary structure of RaCE, we performed site-directed mutagenesis of possible catalytic residues in the enzyme, and the mutants were expressed in Escherichia coli cells. It was found that R52, H243, E246, W249, M251, W304, E308, H374, and M378 were absolutely required for the activity of RaCE. F114 and W303 (and possibly R377) also contributed to catalysis. These possible catalytic protruded into or near the active-site cleft surrounded by the inner α-helices in the predicted (α/α)6 core barrel structure of RaCE.
著者
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森 春英
北海道大学大学院農学研究院応用生命科学部門
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Matsui H
Division Of Applied Bioscience Graduate School Of Agriculture Hokkaido University
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Ito Hiroyuki
Division Of Applied Bioscience Graduate School Of Agriculture Hokkaido University
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Matsui Hirokazu
Dept. Biosci. Chem. Fac. Agric. Hokkaido Univ.
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Matsui Hirokazu
Hokkaido Univ. Sapporo Jpn
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Hiraga Susumu
Department Of Applied Biosciences Graduate School Of Agriculture Hokkaido University
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Ito H
Division Of Applied Bioscience Graduate School Of Agriculture Hokkaido University
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Ito Susumu
Creative Research Initiative Sousei (cris) Hokkaido University
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Ito Shigeaki
Laboratory Of Biochemistry Research Faculty Of Agriculture Hokkaido University
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Hamada Shigeki
Laboratory Of Biochemistry Research Faculty Of Agriculture Hokkaido University
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Matsui Hirokazu
Laboratory Of Biochemistry Research Faculty Of Agriculture Hokkaido University
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Taguchi Hidenori
Laboratory Of Biochemistry Research Faculty Of Agriculture Hokkaido University
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TAGUCHI Hidenori
Research Faculty of Agriculture, Hokkaido University
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