How to design artificial protein surpassing native enzyme function ~ Design and multidisciplinary analysis of artificial L‐threonine 3‐dehydrogenase ~

Abstract

IntroductionCurrently, we can easily obtain a large amount of protein sequence data through database, such as PubMed. The data are being used for protein engineering to reconstruct sequence of target protein, like full‐consensus protein design (FPD) and ancestral sequence reconstruction (ASR). However, redesign of highly functional protein is challenging tasks. In this study, we attempt to get a clue how to succeed in the redesign through protein sequence reconstruction of native l‐threonine 3‐dehydrogenase from Cupriavidus necator (CnTDH) (1).MethodsFirstly, total 5000 of homologous sequences with CnTDH were obtained by submitting sequence of CnTDH to Blastp: the sequences were called “library”. Artificial TDH, FpdTDH‐N0, was designed by the FPD method using the library. Next, utilizing INTMSAlign (2, 3), the curated library was generated to maximize frequency of each consensus residues of artificial TDH. Two artificial TDHs were designed by FPD (FpdTDH‐N1) and ASR (AsrTDH) method, respectively, utilizing the curated library.Results & DiscussionSpecific activity was measured for the three artificial TDHs; and the activity could be confirmed for FpdTDH‐N1 and AsrTDH which designed using the curated library. FpdTDH‐N1 and AsrTDH share 69 and 65% of sequence identity with CnTDH, respectively. Enzyme properties of CnTDH, FpdTDH‐N1 and AsrTDH were estimated by the following multidisciplinary analysis. Thermal stability of both FpdTDH‐N1 and AsrTDH was better than that of CnTDH: Tm values of FpdTDH‐N1 and AsrTDH were higher about 10 and 5 °C than that of CnTDH. Enzyme kinetics parameters of FpdTDH‐N1 and AsrTDH (kcat/Km value) were comparable to CnTDH. Furthermore, dissociation constant (Kd) for AsrTDH and FpdTDH‐N1 toward NAD+ which measured by ITC were about 8‐ and 2‐fold lower than that of CnTDH. Structural comparison among CnTDH, FpdTDH‐N1 (2.9 Å resolution) and ArsTDH (2.2 Å resolution) indicated that there is no mutation around 7 Å of oxygen atom of catalytic Tyr. The mutation was assembled at NAD+ binding site and protein surface.Taken together, we succeeded in design artificial TDHs bearing high stability, activity and affinity toward NAD+ compared with native TDH, CnTDH. This result also inferred that curated library designed by maximizing frequency of amino acids for consensus residues of target protein is effective to design “super enzyme” surpassing native enzyme function.Support or Funding InformationThis work was supported by JSPS KAKENHI Grant Number 16K18688, 17KT0010, 17H06169 and JST by ERATO grant number JPMJER1102.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.