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首页> 外文期刊>Biochemistry >Mutational Effects on Transglycosylating Activity of Family 18 Chitinases and Construction of a Hypertransglycosylating Mutant
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Mutational Effects on Transglycosylating Activity of Family 18 Chitinases and Construction of a Hypertransglycosylating Mutant

机译：突变对家族18几丁质酶的糖基转移活性的影响和一个高糖基化突变体的构建。

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摘要

Enzymatic features that determine transglycosylating activity have been investigated through site-directed mutagenesis studies on two family 18 chitinases, ChiA and ChiB from Serratia marcescens, with inherently little transglycosylation activity. The activity was monitored for the natural substrate (GlcNAc)_4 using mass spectrometry and HPLC. Mutation of the middle Asp in the diagnostic DxDxE motif; which interacts with the catalytic Glu during the catalytic cycle, yielded the strongly transglycosylating mutants ChiA-D313N and ChiB-D142N, respectively. Mutation of the same Asp~(313/142) to Ala or the mutation of Asp~(311/140) to either Asn or Ala had no or much smaller effects on transglycosylating activity. Mutation of Phe~(396) in the +2 subsite of ChiA-D313N to Trp led to a severalfold increase in transglycosylation rate while replacement of aromat,c residues with Ala in the aglycon (sugar acceptor-binding) subsites of ChiA-D313N and ChiB-D142N led to a clear reduction in transglycosylating activity. Taken together, these results show that the transglycosylation properties of family 18 chitinases may be manipulated by mutations that affect the configuration of the catalytic machinery and the affinity for sugar acceptors. The hypertransglycosylating mutant ChiA-D313N-F396W may find applications for synthetic purposes.
机译：通过对诱变粘质沙雷氏菌的两个家族18几丁质酶ChiA和ChiB进行定点诱变研究，研究了确定转糖基化活性的酶学特征，而固有的转糖基化活性却很小。使用质谱和HPLC监测天然底物（GlcNAc）_4的活性。诊断性DxDxE基序中中间Asp的突变;在催化循环过程中与催化Glu相互作用的Aβ分别产生强转糖基化的突变体ChiA-D313N和ChiB-D142N。相同的Asp〜（313/142）突变为Ala或Asp〜（311/140）突变为Asn或Ala对转糖基化活性没有影响或影响很小。 ChiA-D313N +2亚位点的Phe〜（396）突变为Trp导致转糖基化速率提高了几倍，而ChiA-D313N和糖苷结合位点的糖苷结合亚位点上的芳香残基被Ala取代。 ChiB-D142N导致转糖基化活性明显降低。综上所述，这些结果表明，家族18几丁质酶的转糖基化特性可通过影响催化机制的构型和对糖受体亲和力的突变来操纵。高反糖基化的突变体ChiA-D313N-F396W可以用于合成目的。

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