5Z76

Artificial L-threonine 3-dehydrogenase designed by full consensus design

Summary for 5Z76

• Entry DOI: 10.2210/pdb5z76/pdb
• Descriptor: Artificial L-threonine 3-dehydrogenase (1 entity in total)
• Functional Keywords: l-threonine 3-dehydrogenase, full consensus design, oxidoreductase
• Biological source: synthetic construct
• Total number of polymer chains: 4
• Total formula weight: 151636.62

Authors

Nakano, S.,Motoyama, T.,Miyashita, Y.,Ishizuka, Y.,Matsuo, N.,Tokiwa, H.,Shinoda, S.,Asano, Y.,Ito, S. (deposition date: 2018-01-27, release date: 2018-08-22, Last modification date: 2023-11-22)

Primary citation

Nakano, S.,Motoyama, T.,Miyashita, Y.,Ishizuka, Y.,Matsuo, N.,Tokiwa, H.,Shinoda, S.,Asano, Y.,Ito, S.
Benchmark Analysis of Native and Artificial NAD+-Dependent Enzymes Generated by a Sequence-Based Design Method with or without Phylogenetic Data.
Biochemistry, 57:3722-3732, 2018

PubMed 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 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 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.

PubMed: 29787243
DOI: 10.1021/acs.biochem.8b00339

Experimental method

X-RAY DIFFRACTION (2.8 Å)