Fujita K, Takegawa K
Department of Life Sciences, Kagawa University, Miki-cho, Kagawa, 761-0795, Japan.
Biochem Biophys Res Commun. 2001 May 11;283(3):680-6. doi: 10.1006/bbrc.2001.4836.
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.
来自原光节杆菌的内切-β-N-乙酰氨基葡萄糖苷酶(Endo-A)具有高水平的转糖基化活性。为了确定哪些氨基酸参与了这种活性,我们采用了缺失分析以及随机诱变和定点诱变。通过PCR随机诱变,分离出了11个酶活性水平大幅降低的突变体。通过定点诱变鉴定出了6个催化必需氨基酸。突变体E173G、E175Q、D206G和D270N的水解活性显著降低,而突变体V109D、E173D和E173Q失去了所有酶活性,这表明Val-109和Glu-173对催化功能很重要。此外,我们分离出了一个随机突变,该突变消除了转糖基化活性但不影响水解活性。通过定点诱变确定,Trp-216突变为Arg是导致转糖基化活性丧失的原因。虽然Trp-216的其他突变体活性降低,但突变为另一个带正电荷的残基(Lys)也消除了转糖基化活性。与最近克隆的另外两种具有转糖基化活性的内切-β-N-乙酰氨基葡萄糖苷酶进行序列比较,发现这三种酶的N端区域具有高度同源性。这些结果表明,Endo-A第216位的色氨酸残基在转糖基化中起关键作用。