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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.

Authors :
Nakano S
Motoyama T
Miyashita Y
Ishizuka Y
Matsuo N
Tokiwa H
Shinoda S
Asano Y
Ito S
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.

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