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Benchmark Analysis of Native and Artificial NAD + -Dependent Enzymes Generated by a Sequence-Based Design Method with or without Phylogenetic Data.
- Source :
-
Biochemistry [Biochemistry] 2018 Jul 03; Vol. 57 (26), pp. 3722-3732. Date of Electronic Publication: 2018 Jun 04. - Publication Year :
- 2018
-
Abstract
- The expansion of protein sequence databases has enabled us to design artificial proteins by sequence-based design methods, such as full-consensus design (FCD) and ancestral-sequence reconstruction (ASR). Artificial proteins with enhanced activity levels compared with native ones can potentially be generated by such methods, but successful design is rare because preparing a sequence library by curating the database and selecting a method is difficult. Utilizing a curated library prepared by reducing conservation energies, we successfully designed two artificial l-threonine 3-dehydrogenases (SDR-TDH) with higher activity levels than native SDR-TDH, FcTDH-N1, and AncTDH, using FCD and ASR, respectively. The artificial SDR-TDHs had excellent thermal stability and NAD <superscript>+</superscript> recognition compared to native SDR-TDH from Cupriavidus necator (CnTDH); the melting temperatures of FcTDH-N1 and AncTDH were about 10 and 5 °C higher than that of CnTDH, respectively, and the dissociation constants toward NAD <superscript>+</superscript> of FcTDH-N1 and AncTDH were 2- and 7-fold lower than that of CnTDH, respectively. Enzymatic efficiency of the artificial SDR-TDHs were comparable to that of CnTDH. Crystal structures of FcTDH-N1 and AncTDH were determined at 2.8 and 2.1 Å resolution, respectively. Structural and MD simulation analysis of the SDR-TDHs indicated that only the flexibility at specific regions was changed, suggesting that multiple mutations introduced in the artificial SDR-TDHs altered their flexibility and thereby affected their enzymatic properties. Benchmark analysis of the SDR-TDHs indicated that both FCD and ASR can generate highly functional proteins if a curated library is prepared appropriately.
- Subjects :
- Alcohol Oxidoreductases chemistry
Alcohol Oxidoreductases genetics
Amino Acid Sequence
Biotechnology methods
Crystallography, X-Ray
Cupriavidus necator chemistry
Cupriavidus necator genetics
Cupriavidus necator metabolism
Enzyme Stability
Models, Molecular
Phylogeny
Plasmids genetics
Protein Engineering methods
Protein Folding
Substrate Specificity
Alcohol Oxidoreductases metabolism
Cupriavidus necator enzymology
NAD metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1520-4995
- Volume :
- 57
- Issue :
- 26
- Database :
- MEDLINE
- Journal :
- Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 29787243
- Full Text :
- https://doi.org/10.1021/acs.biochem.8b00339