Structural insights on mouse l-threonine dehydrogenase: A regulatory role of Arg180 in catalysis

• Published in: Journal of structural biology Vol. 192; no. 3; pp. 510 - 518
• Main Authors: He, Chao, Huang, Xianyu, Liu, Yanhong, Li, Fudong, Yang, Yang, Tao, Hongru, Han, Chuanchun, Zhao, Chen, Xiao, Yazhong, Shi, Yunyu
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.12.2015
• Subjects: Alcohol Oxidoreductases - chemistry; Alcohol Oxidoreductases - genetics; Alcohol Oxidoreductases - ultrastructure; Amino Acid Sequence; Animals; Arg180; Binding Sites; Calorimetry; Catalysis; Crystal structure; Crystallography, X-Ray; Dehydrogenase; Enzyme kinetics; Enzyme mutation; Glycerol - chemistry; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; l-Threonine; Mice; Models, Molecular; Molecular Sequence Data; Mouse; Mutation - genetics; NAD - chemistry; Nicotinamide adenine dinucleotide (NAD); Substrate Specificity; Threonine - chemistry; SDR; PRMT5; ASU; Nicotinamide adenine dinucleotide (NAD); Dehydrogenase; Enzyme mutation; GalE; Arg180; Mouse; TDH; mTDH; l-Thr; mESC; MDR; l-Threonine; Enzyme kinetics; WT; Crystal structure
• ISSN: 1047-8477; 1095-8657
• DOI: 10.1016/j.jsb.2015.10.014

Mouse l-threonine dehydrogenase (mTDH), which belongs to the short-chain dehydrogenase/reductase (SDR) superfamily and mediates threonine catabolism, plays pivotal roles in both powerful biosynthesis and signaling in mouse stem cells and has a regulatory residue Arg180. Here we determined three crystal structures of mTDH: wild-type (WT) in the apo form; in complex with NAD+ and a substrate analog, glycerol, or with only NAD+; as well as the R180K variant with NAD+. This is the first description of a structure for mammalian SDR-type TDH. Structural comparison revealed the structural basis for SDR-type TDH catalysis remains strictly conserved in bacteria and mammals. Kinetic enzyme assays, and isothermal titration calorimetry (ITC) measurements indicated the R180K mutation has little effect on NAD+ binding affinity, whereas affects the substrate’s affinity for the enzyme. The crystal structure of R180K with NAD+, biochemical and spectroscopic studies suggested that the R180K mutant should bind NAD+ in a similar way and have a similar folding to the WT. However, the R180K variant may have difficulty adopting the closed form due to reduced interaction of residue 180 with a loop which connects a key position for mTDH switching between the closed and open forms in mTDH catalysis, and thereby exhibited a significantly decreased kcat/Km value toward the substrate, l-Thr. In sum, our results suggest that activity of GalE-like TDH can be regulated by remote interaction, such as hydrogen bonding and hydrophobic interaction around the Arg180 of mTDH.