Tryptophan-216 is essential for the transglycosylation activity of endo-beta-N-acetylglucosaminidase A.
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 recently, reveals a high degree of identity in the N-terminal regions of the three enzymes. These results indicate that the tryptophan residue at position 216 of Endo-A has a key role in the transglycosylation.
Fujita
K
K
Department of Life Sciences, Kagawa University, Miki-cho, Kagawa, 761-0795, Japan.
Takegawa
K
K
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001173:Arthrobacter / Q000201:enzymology / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D006031:Glycosylation; D006868:Hydrolysis; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D014364:Tryptophan / Q000737:chemistry
D014364:Tryptophan; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase