Department of Chemistry Biotechnology and Food Science, Norwegian University of Life Sciences, P.O. Box 5003, N-1432 Aas, Norway.
Biochemistry. 2011 Jun 28;50(25):5693-703. doi: 10.1021/bi2002532. Epub 2011 Jun 2.
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 aromatic 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.
通过对具有天然低转糖苷活性的沙雷氏菌属丝氨酸 18 家族几丁质酶 ChiA 和 ChiB 的定点突变研究，探讨了决定转糖苷活性的酶学特性。利用质谱和 HPLC 监测天然底物（GlcNAc）（4）的活性。突变催化循环中与催化Glu 相互作用的诊断性 DxDxE 基序中的中间 Asp，分别得到了强转糖苷突变体 ChiA-D313N 和 ChiB-D142N。将相同的 Asp（313/142）突变为 Ala 或 Asp（311/140）突变为 Asn 或 Ala，对转糖苷活性没有影响或影响较小。将 ChiA-D313N 中的+2 亚基的 Phe（396）突变为 Trp，导致转糖苷速率增加几倍，而在 ChiA-D313N 和 ChiB-D142N 的非糖（糖受体结合）亚基中用 Ala 替换芳香残基，则明显降低了转糖苷活性。总之，这些结果表明，突变可以改变催化机制的构象和对糖受体的亲和力，从而改变 18 家族几丁质酶的转糖苷特性。高转糖苷突变体 ChiA-D313N-F396W 可能具有合成用途。
