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Enhanced Azidolysis by the Formation of Stable Ser–His Catalytic Dyad in a Glycoside Hydrolase Family 10 Xylanase Mutant by Ryuichiro Suzuki; Zui Fujimoto; Satoshi Kaneko; Tsunemi Hasegawa; Atsushi Kuno is a Biochemistry, Genetics and Molecular Biology article available to read on EtoBox.

What is Enhanced Azidolysis by the Formation of Stable Ser–His Catalytic Dyad in a Glycoside Hydrolase Family 10 Xylanase Mutant about?

Glycoside hydrolases require carboxyl groups as catalysts for their activity. A retaining xylanase from __Streptomyces olivaceoviridis__ E-86 belonging to glycoside hydrolase family 10 possesses Glu128 and Glu236 that respectively function as acid/base and nucleophile. We previously developed a unique mutant of the retaining xylanase, N127S/E128H, whose deglycosylation is triggered by azide. A crystallographic study reported that the transient formation of a Ser–His catalytic dyad in the reaction cycle possibly reduced the azidolysis reaction. In the present study, we engineered a catalytic dyad with enhanced stability by site-directed mutagenesis and crystallographic study of N127S/E128H. Comparison of the Michaelis complexes of N127S/E128H with pNP-X~2~ and with xylopentaose showed that Ser127 could form an alternative hydrogen bond with Thr82, which disrupts the formation of the Ser–His catalytic dyad. The introduction of T82A mutation in N127S/E128H produces an enhanced first-order rate constant (6 times that of N127S/E128H). We confirmed the presence of a stable Ser–His hydrogen bond in the Michaelis complex of the triple mutant, which forms the productive tautomer of His128 t

Who reads Enhanced Azidolysis by the Formation of Stable Ser–His Catalytic Dyad in a Glycoside Hydrolase Family 10 Xylanase Mutant?

Author

Ryuichiro Suzuki; Zui Fujimoto; Satoshi Kaneko; Tsunemi Hasegawa; Atsushi Kuno

Publisher

The Japanese Society of Applied Glycoscience

Published

2018

Language

EN

Field

Biochemistry, Genetics and Molecular Biology (Life Sciences)