5z76

Publication Abstract from PubMed

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 degrees 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 A 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.

Benchmark Analysis of Native and Artificial NAD(+)-Dependent Enzymes Generated by a Sequence-Based Design Method with or without Phylogenetic Data.,Nakano S, Motoyama T, Miyashita Y, Ishizuka Y, Matsuo N, Tokiwa H, Shinoda S, Asano Y, Ito S Biochemistry. 2018 Jul 3;57(26):3722-3732. doi: 10.1021/acs.biochem.8b00339. Epub, 2018 Jun 4. PMID:29787243^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.