Structural insights on mouse L-threonine dehydrogenase: A regulatory role of Arg180 in catalysis.
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.
Anhui Provincial Engineering Technology Research Center of Microorganisms and Biocatalysis and School of Life Sciences, Anhui University, Hefei, Anhui 230601, China. Electronic address: chaohe@ahu.edu.cn.
Chao
C
He
Anhui Provincial Engineering Technology Research Center of Microorganisms and Biocatalysis and School of Life Sciences, Anhui University, Hefei, Anhui 230601, China.
Xianyu
X
Huang
Anhui Provincial Engineering Technology Research Center of Microorganisms and Biocatalysis and School of Life Sciences, Anhui University, Hefei, Anhui 230601, China.
Yanhong
Y
Liu
Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, China.
Fudong
F
Li
Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, China.
Yang
Y
Yang
Anhui Provincial Engineering Technology Research Center of Microorganisms and Biocatalysis and School of Life Sciences, Anhui University, Hefei, Anhui 230601, China.
Hongru
H
Tao
Institute of Cancer Stem Cell, Dalian Medical University, Dalian, Liaoning 116044, China.
Chuanchun
C
Han
Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, China.
Chen
C
Zhao
Anhui Provincial Engineering Technology Research Center of Microorganisms and Biocatalysis and School of Life Sciences, Anhui University, Hefei, Anhui 230601, China.
Yazhong
Y
Xiao
Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, China.
Yunyu
Y
Shi
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002151:Calorimetry; D002384:Catalysis; D018360:Crystallography, X-Ray; D005990:Glycerol / Q000737:chemistry*; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009243:NAD / Q000737:chemistry*; D013379:Substrate Specificity; D013912:Threonine / Q000737:chemistry*
D009243:NAD; D013912:Threonine; D000429:Alcohol Oxidoreductases; C023159:L-threonine 3-dehydrogenase; D005990:Glycerol
Arg180; Crystal structure; Dehydrogenase; Enzyme kinetics; Enzyme mutation; Mouse; Nicotinamide adenine dinucleotide (NAD); l-Threonine