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bioRxiv · 10.1101/2021.11.25.470051

Genetic perturbation enhances functional heterogeneity in alkaline phosphatase

Sakuma, M.·Honda, S.·Ueno, H.·Miyazaki, K.·Tokuriki, N.·Noji, H.
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Abstract

Enzymes inherently exhibit molecule-to-molecule heterogeneity in catalytic activity or function, which underlies the acquisition of new functions in evolutionary processes. However, correlations between the functional heterogeneity of an enzyme and its multi-functionality or promiscuity remain elusive. In addition, the modulation of functional heterogeneity upon genetic perturbation is currently unexplored. Here, we quantitatively analyzed functional heterogeneity in the wild-type and 69 single-point mutants of Escherichia coli alkaline phosphatase (AP) by employing single-molecule assay with a femtoliter reactor array device. Most mutant enzymes exhibited higher functional heterogeneity than the wild-type enzyme, irrespective of catalytic activity. These results indicated that the wild-type AP minimizes functional heterogeneity, and single-point mutations can significantly expand the span of functional heterogeneity in AP. Moreover, we identified a clear correlation between functional heterogeneity and promiscuous activities. These findings suggest that enzymes can acquire greater functional heterogeneity following marginal genetic perturbations that concomitantly promote catalytic promiscuity.

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Sakuma, M., Honda, S., Ueno, H., Miyazaki, K., Tokuriki, N., Noji, H.. 2021-11-25. Genetic perturbation enhances functional heterogeneity in alkaline phosphatase. https://doi.org/10.1101/2021.11.25.470051

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