3VPH

L-lactate dehydrogenase from Thermus caldophilus GK24 complexed with oxamate, NADH and FBP

Summary for 3VPH

• Entry DOI: 10.2210/pdb3vph/pdb
• Related: 3VPG
• Descriptor: L-lactate dehydrogenase, OXAMIC ACID, 1,6-di-O-phosphono-beta-D-fructofuranose, ... (6 entities in total)
• Functional Keywords: rossmann fold, dehydrogenase, oxidoreductase
• Biological source: Thermus caldophilus
• Cellular location: Cytoplasm: P06150
• Total number of polymer chains: 4
• Total formula weight: 134673.39

Authors

Arai, K.,Ohno, T.,Miyanaga, A.,Fushinobu, S.,Taguchi, H. (deposition date: 2012-03-01, release date: 2013-03-06, Last modification date: 2023-11-08)

Primary citation

Ikehara, Y.,Arai, K.,Furukawa, N.,Ohno, T.,Miyake, T.,Fushinobu, S.,Nakajima, M.,Miyanaga, A.,Taguchi, H.
The core of allosteric motion in Thermus caldophilus L-lactate dehydrogenase.
J.Biol.Chem., 2014

PubMed Abstract: For Thermus caldophilus L-lactate dehydrogenase (TcLDH), fructose 1,6-bisphosphate (FBP) reduced the pyruvate S(0.5) value 10(3)-fold and increased the V(max) value 4-fold at 30 °C and pH 7.0, indicating that TcLDH has a much more T state-sided allosteric equilibrium than Thermus thermophilus L-lactate dehydrogenase, which has only two amino acid replacements, A154G and H179Y. The inactive (T) and active (R) state structures of TcLDH were determined at 1.8 and 2.0 Å resolution, respectively. The structures indicated that two mobile regions, MR1 (positions 172-185) and MR2 (positions 211-221), form a compact core for allosteric motion, and His(179) of MR1 forms constitutive hydrogen bonds with MR2. The Q4(R) mutation, which comprises the L67E, H68D, E178K, and A235R replacements, increased V(max) 4-fold but reduced pyruvate S(0.5) only 5-fold in the reaction without FBP. In contrast, the P2 mutation, comprising the R173Q and R216L replacements, did not markedly increase V(max), but 10(2)-reduced pyruvate S(0.5), and additively increased the FBP-independent activity of the Q4(R) enzyme. The two types of mutation consistently increased the thermal stability of the enzyme. The MR1-MR2 area is a positively charged cluster, and its center approaches another positively charged cluster (N domain cluster) across the Q-axis subunit interface by 5 Å, when the enzyme undergoes the T to R transition. Structural and kinetic analyses thus revealed the simple and unique allosteric machinery of TcLDH, where the MR1-MR2 area pivotally moves during the allosteric motion and mediates the allosteric equilibrium through electrostatic repulsion within the protein molecule.

PubMed: 25258319
DOI: 10.1074/jbc.M114.599092

Experimental method

X-RAY DIFFRACTION (2 Å)