Genetic Perturbation Alters Functional Substates in Alkaline Phosphatase
Enzymes inherently exhibit molecule-to-molecule heterogeneity in their conformational and functional states, which is considered to be a key to the evolution of new functions. Single-molecule enzyme assays enable us to directly observe such multiple functional states or functional substates. Here, w...
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| Veröffentlicht in: | Journal of the American Chemical Society 2023-02, Vol.145 (5), p.2806-2814 |
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| container_title | Journal of the American Chemical Society |
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| creator | Sakuma, Morito Honda, Shingo Ueno, Hiroshi Tabata, Kazuhito V. Miyazaki, Kentaro Tokuriki, Nobuhiko Noji, Hiroyuki |
| description | Enzymes inherently exhibit molecule-to-molecule heterogeneity in their conformational and functional states, which is considered to be a key to the evolution of new functions. Single-molecule enzyme assays enable us to directly observe such multiple functional states or functional substates. Here, we quantitatively analyzed functional substates in the wild-type and 69 single-point mutants of Escherichia coli alkaline phosphatase by employing a high-throughput single-molecule assay with a femtoliter reactor array device. Interestingly, many mutant enzymes exhibited significantly heterogeneous functional substates with various types, while the wild-type enzyme showed a highly homogeneous substate. We identified a correlation between the degree of functional substates and the level of improvement in promiscuous activities. Our work provides much comprehensive evidence that the functional substates can be easily altered by mutations, and the evolution toward a new catalytic activity may involve the modulation of the functional substates. |
| doi_str_mv | 10.1021/jacs.2c06693 |
| format | Article |
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Single-molecule enzyme assays enable us to directly observe such multiple functional states or functional substates. Here, we quantitatively analyzed functional substates in the wild-type and 69 single-point mutants of Escherichia coli alkaline phosphatase by employing a high-throughput single-molecule assay with a femtoliter reactor array device. Interestingly, many mutant enzymes exhibited significantly heterogeneous functional substates with various types, while the wild-type enzyme showed a highly homogeneous substate. We identified a correlation between the degree of functional substates and the level of improvement in promiscuous activities. 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| source | MEDLINE; ACS Publications |
| subjects | Alkaline Phosphatase - chemistry Alkaline Phosphatase - genetics Escherichia coli - genetics Escherichia coli Proteins - chemistry Escherichia coli Proteins - genetics Mutation Protein Conformation |
| title | Genetic Perturbation Alters Functional Substates in Alkaline Phosphatase |
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