Active and inactive state structures of unliganded Lactobacillus casei allosteric L-lactate dehydrogenase

Proteins. 2010 Feb 15;78(3):681-94. doi: 10.1002/prot.22597.

Abstract

Lactobacillus casei L-lactate dehydrogenase (LCLDH) is activated through the homotropic and heterotropic activation effects of pyruvate and fructose 1,6-bisphosphate (FBP), respectively, and exhibits unusually high pH-dependence in the allosteric effects of these ligands. The active (R) and inactive (T) state structures of unliganded LCLDH were determined at 2.5 and 2.6 A resolution, respectively. In the catalytic site, the structural rearrangements are concerned mostly in switching of the orientation of Arg171 through the flexible intersubunit contact at the Q-axis subunit interface. The distorted orientation of Arg171 in the T state is stabilized by a unique intra-helix salt bridge between Arg171 and Glu178, which is in striking contrast to the multiple intersubunit salt bridges in Lactobacillus pentosus nonallosteric L-lactate dehydrogenase. In the backbone structure, major structural rearrangements of LCLDH are focused in two mobile regions of the catalytic domain. The two regions form an intersubunit linkage through contact at the P-axis subunit interface involving Arg185, replacement of which with Gln severely decreases the homotropic and hetertropic activation effects on the enzyme. These two regions form another intersubunit linkage in the Q-axis related dimer through the rigid NAD-binding domain, and thus constitute a pivotal frame of the intersubunit linkage for the allosteric motion, which is coupled with the concerted structural change of the four subunits in a tetramer, and of the binding sites for pyruvate and FBP. The unique intersubunit salt bridges, which are observed only in the R state structure, are likely involved in the pH-dependent allosteric equilibrium.

MeSH terms

  • Amino Acid Sequence
  • Catalytic Domain
  • Crystallography, X-Ray
  • Fructose-Bisphosphatase / chemistry
  • Fructose-Bisphosphatase / metabolism
  • L-Lactate Dehydrogenase / chemistry*
  • L-Lactate Dehydrogenase / genetics
  • L-Lactate Dehydrogenase / metabolism
  • Lacticaseibacillus casei / enzymology*
  • Models, Molecular
  • Molecular Sequence Data
  • Mutation
  • Protein Binding
  • Protein Conformation
  • Sequence Alignment

Substances

  • L-Lactate Dehydrogenase
  • Fructose-Bisphosphatase