Tryptophan-216 is essential for the transglycosylation activity of endo-beta-N-acetylglucosaminidase A

Biochemical and Biophysical Research Communications
K Fujita, K Takegawa

Abstract

Endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) has a high level of transglycosylation activity. To determine which amino acids are involved in this activity, we employed deletion analysis, as well as random and site-directed mutagenesis. Using PCR random mutagenesis, 11 mutants with greatly decreased levels of enzyme activity were isolated. Six catalytically essential amino acids were identified by site-directed mutagenesis. Mutants E173G, E175Q, D206G, and D270N had markedly reduced hydrolysis activity, while mutants V109D, E173D, and E173Q lost all enzymatic activity, indicating that Val-109 and Glu-173 are important for the catalytic function. Moreover, we isolated a random mutation that abolished the transglycosylation activity without affecting the hydrolysis activity. The Trp-216 to Arg mutation was identified, by site-directed mutagenesis, as that responsible for the loss of transglycosylation activity. While other mutants of Trp-216 showed reduced activity, mutation to another positively charged residue (Lys) also abolished the transglycosylation activity. Sequence comparison with two other endo-beta-N-acetylglucosaminidases, that possess transglycosylation activity and that have been cloned ...Continue Reading

References

Feb 1, 1979·Analytical Biochemistry·E W Holmes, J S O'Brien
Feb 1, 1992·Yeast·D MuhlradR Parker
Nov 15, 1991·European Journal of Biochemistry·K TakegawaT Tochikura
Dec 1, 1986·Journal of Bacteriology·H SuzukiT Tochikura
Dec 14, 1999·Protein Science : a Publication of the Protein Society·V RaoP Van Roey
Jan 7, 2000·Biochemical and Biophysical Research Communications·K FujitaK Takegawa

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Citations

Jun 1, 2004·Bioscience, Biotechnology, and Biochemistry·Kiyotaka FujitaKaoru Takegawa
Mar 20, 2009·Nature Chemical Biology·Jamie R Rich, Stephen G Withers
Aug 5, 2008·Chembiochem : a European Journal of Chemical Biology·Christoph D HeideckeAntony J Fairbanks
Aug 30, 2008·The New Phytologist·Lindsey SchroevenWim Van den Ende
Oct 15, 2009·Chemical Reviews·Shiro Kobayashi, Akira Makino
Nov 3, 2004·Archives of Biochemistry and Biophysics·Kiyotaka FujitaKenji Yamamoto

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