Journal
NUCLEIC ACIDS RESEARCH
Volume 50, Issue 19, Pages 11175-11185Publisher
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkac858
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Funding
- US National Institutes of Health [GM108703]
- Simons Foundation [340762]
- National Aeronautics and Space Administration [80NSSC21K0595]
- Minnesota Medical Foundation [4036-9663-10]
- University of Minnesota Biocatalysis Initiative
- Office of the VP of Research at the University of Minnesota
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In this study, the catalytic activity of an artificial enzyme was improved by fusing different protein domains, demonstrating that domain fusion mechanism in nature can enhance the functionality of newly created proteins.
The function of most proteins is accomplished through the interplay of two or more protein domains and fine-tuned by natural evolution. In contrast, artificial enzymes have often been engineered from a single domain scaffold and frequently have lower catalytic activity than natural enzymes. We previously generated an artificial enzyme that catalyzed an RNA ligation by >2 million-fold but was likely limited in its activity by low substrate affinity. Inspired by nature's concept of domain fusion, we fused the artificial enzyme to a series of protein domains known to bind nucleic acids with the goal of improving its catalytic activity. The effect of the fused domains on catalytic activity varied greatly, yielding severalfold increases but also reductions caused by domains that previously enhanced nucleic acid binding in other protein engineering projects. The combination of the two better performing binding domains improved the activity of the parental ligase by more than an order of magnitude. These results demonstrate for the first time that nature's successful evolutionary mechanism of domain fusion can also improve an unevolved primordial-like protein whose structure and function had just been created in the test tube. The generation of multi-domain proteins might therefore be an ancient evolutionary process.
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