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Benchmark Analysis of Native and Artificial NAD+-Dependent Enzymes Generated by a Sequence-Based Design Method with
Shogo Nakano1,2, Tomoharu Motoyama1, Yurina Miyashita3
1Graduate Division of Nutritional and Environmental Sciences , University of Shizuoka , 52-1 Yada , Suruga-ku, Shizuoka 422-8526 , Japan.
Biochemistry
|May 23, 2018
Summary
Researchers designed artificial l-threonine 3-dehydrogenases (SDR-TDH) with enhanced activity and stability using full-consensus design (FCD) and ancestral-sequence reconstruction (ASR) methods. These artificial proteins demonstrate improved thermal stability and cofactor binding, showcasing the potential of curated libraries for protein engineering.
Area of Science:
- Protein Engineering
- Computational Biology
- Biochemistry
Background:
- Protein sequence databases facilitate artificial protein design via methods like full-consensus design (FCD) and ancestral-sequence reconstruction (ASR).
- Achieving enhanced protein activity is challenging due to difficulties in curating sequence libraries and selecting appropriate design methods.
- Native l-threonine 3-dehydrogenases (SDR-TDH) serve as a benchmark for evaluating engineered variants.
Purpose of the Study:
- To design and characterize novel artificial SDR-TDH variants with improved properties compared to native enzymes.
- To evaluate the efficacy of FCD and ASR methods when applied to a curated sequence library.
- To elucidate the structural and dynamic basis for enhanced enzyme function in engineered proteins.
Main Methods:
- Utilized a curated sequence library derived from reducing conservation energies.
- Employed FCD and ASR to generate artificial SDR-TDH variants (FcTDH-N1 and AncTDH).
- Characterized enzyme activity, thermal stability, NAD+ binding affinity, and crystal structures; performed molecular dynamics (MD) simulations.
Main Results:
- Successfully designed two artificial SDR-TDHs, FcTDH-N1 and AncTDH, exhibiting higher activity than native SDR-TDH.
- FcTDH-N1 and AncTDH demonstrated superior thermal stability (10°C and 5°C higher melting temperatures, respectively) and NAD+ recognition (2- and 7-fold lower dissociation constants, respectively) compared to native CnTDH.
- Enzymatic efficiency was comparable to native enzymes, with structural and MD analyses revealing altered flexibility in specific regions due to mutations.
Conclusions:
- Both FCD and ASR, when utilizing an appropriately curated library, can successfully generate highly functional artificial proteins.
- The study highlights the importance of sequence library curation in achieving successful protein design outcomes.
- Engineered changes in protein flexibility significantly influence enzymatic properties, offering insights into protein engineering strategies.
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