Substrate-Binding Mode and Intermediate-Product Distribution Coguided Protein Design of Alginate Lyase AlyF for Altered End-Product Distribution
| Autor: | Yan Yang, Weizhi Liu, Qianqian Lyu, Weidong Wang, Keke Zhang |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
0106 biological sciences
chemistry.chemical_classification Stereochemistry 010401 analytical chemistry Mutant Protein design Disaccharide Substrate (chemistry) General Chemistry Protein engineering 01 natural sciences Intermediate product Product distribution 0104 chemical sciences chemistry.chemical_compound chemistry Trisaccharide General Agricultural and Biological Sciences 010606 plant biology & botany |
| Zdroj: | Journal of Agricultural and Food Chemistry. 69:7190-7198 |
| ISSN: | 1520-5118 0021-8561 |
| DOI: | 10.1021/acs.jafc.1c02473 |
| Popis: | Recently, we reported alginate lyase AlyF that predominantly produced trisaccharides (the trisaccharide content is 87.0%), and the determination of its substrate-binding mode facilitated its protein engineering for new product distribution. To clarify the relationship between the substrate-binding pocket and end-product distribution, the open binding pocket change was initially designed. The resulting F128T_W172R mutant of AlyF exhibited different intermediate-product distributions but still similar end-product distributions. However, these observations suggested that cleavage pattern changes for intermediate products might contribute to an altered end-product distribution. Structural analysis indicated that the sugar-binding affinity at subsite -2 should be redesigned to achieve this goal. Thus, residue Arg266, which is involved in sugar binding at subsite -2, was selected for site-saturation mutagenesis in the F128T_W172R mutant. The dominant end products of the F128T_W172R_R226H mutant were altered to disaccharides and trisaccharides (the disaccharide content increased to 40.5%). |
| Databáze: | OpenAIRE |
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