IntroductionL‐Threonine (L‐Thr) metabolism is not well studied, however, plays some part in the lipid synthesis of Trypanosoma brucei and the growth of mouse embryonic stem (ES) cells. L‐Thr is initially catalyzed by L‐threonine dehydrogenase (TDH) which is a NAD+‐dependent enzyme catalyzing dehydrogenation of hydroxyl group of L‐Thr to 2‐amino 3‐ketobutyrate (AKB). TDH belonging to short chain dehydrogenase/reductase family has high specificity toward L‐Thr; the specificity is regulated by switching between open state and closed state by binding of substrate (1). On the other hand, the product release mechanism remains to be elucidated. In this study, we discuss the reaction mechanism associated with structural change at the active site by combinational analysis, such as high resolution X‐ray crystal structure, quantum mechanical (QM) calculation method and kinetic analysis.MethodsIn silico screening was performed using INTMSAlign (2) to obtain TDH (mtTDH) derived from metagenome library. At first, we evaluated enzymatic properties of mtTDH. Four crystal structures of mtTDH were determined at 1.25 – 1.9 Å resolution: apo, binary (NAD+‐binding) and two ternary (l‐Ser‐NAD+‐binding and AKB‐NADH‐binding). For the AKB‐NADH‐binding structure, the Inter‐Fragment Interaction Energy (IFIE) were calculated by the Fragment Molecular Orbital (FMO) method. We also performed site‐directed mutagenesis (S74A, S111A, Y136F, T177A, D179A, and D179N) and kinetic studies to elucidate the roles of active site residues.Results & DiscussionThe crystal structure of mtTDH was obtained as a monomeric form; this is consistent with the result of gel‐filtration chromatography. Based on these results, we concluded that mtTDH is monomeric TDH. Structural comparison of the four structures revealed that ligand (AKB or l‐Thr) and eight molecules (six residues and two molecules of water) appeared to form hydrogen bonding interactions each other. In order to evaluate these interactions quantitatively, IFIEs were calculated by FMO method which is a kind of QM calculation method. As a result, surprisingly, D179 had a repulsive interaction with the AKB. Kinetic analysis of mtTDH variants, D179A and D179N, exhibited the remarkably decrease in activity in spite that D179 works against formation of interaction with the ligand. Taken together, D179 may be a sensor detecting the completion of the reaction and important for switching between open state and closed state (3).Support or Funding InformationThis work was supported by JSPS KAKENHI Grants 16K18688, 17KT0010, and 17K06931 and JST by ERATO Grant 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.