Insights into the molecular relationships between malate and lactate dehydrogenases: structural and biochemical properties of monomeric and dimeric intermediates of a mutant of tetrameric L-[LDH-like] malate dehydrogenase from the halophilic archaeon Haloarcula marismortui

Biochemistry. 2000 Feb 8;39(5):1001-10. doi: 10.1021/bi9910023.

Abstract

L-Malate (MalDH) and L-lactate (LDH) dehydrogenases belong to the same family of NAD-dependent enzymes. LDHs are tetramers, whereas MalDHs can be either dimeric or tetrameric. To gain insight into molecular relationships between LDHs and MalDHs, we studied folding intermediates of a mutant of the LDH-like MalDH (a protein with LDH-like structure and MalDH enzymatic activity) from the halophilic archaeon Haloarcula marismortui (Hm MalDH). Crystallographic analysis of Hm MalDH had shown a tetramer made up of two dimers interacting mainly via complex salt bridge clusters. In the R207S/R292S Hm MalDH mutant, these salt bridges are disrupted. Its structural parameters, determined by neutron scattering and analytical centrifugation under different conditions, showed the protein to be a tetramer in 4 M NaCl. At lower salt concentrations, stable oligomeric intermediates could be trapped at a given pH, temperature, or NaCl solvent concentration. The spectroscopic properties and enzymatic behavior of monomeric, dimeric, and tetrameric species were thus characterized. The properties of the dimeric intermediate were compared to those of dimeric intermediates of LDH and dimeric MalDHs. A detailed analysis of the putative dimer-dimer contact regions in these enzymes provided an explanation of why some can form tetramers and others cannot. The study presented here makes Hm MalDH the best characterized example so far of an LDH-like MalDH.

Publication types

  • Comparative Study

MeSH terms

  • Anilino Naphthalenesulfonates / chemistry
  • Arginine / genetics
  • Dimerization
  • Enzyme Activation / genetics
  • Enzyme Stability / genetics
  • Haloarcula marismortui / enzymology*
  • Haloarcula marismortui / genetics
  • Hydrogen-Ion Concentration
  • L-Lactate Dehydrogenase / chemistry*
  • L-Lactate Dehydrogenase / genetics*
  • L-Lactate Dehydrogenase / metabolism
  • Malate Dehydrogenase / chemistry*
  • Malate Dehydrogenase / genetics*
  • Malate Dehydrogenase / metabolism
  • Mutagenesis, Site-Directed*
  • Neutrons
  • Point Mutation
  • Protein Structure, Secondary / genetics
  • Scattering, Radiation
  • Serine / genetics
  • Sodium Chloride / chemistry
  • Spectrometry, Fluorescence
  • Ultracentrifugation

Substances

  • Anilino Naphthalenesulfonates
  • Sodium Chloride
  • Serine
  • 1-anilino-8-naphthalenesulfonate
  • Arginine
  • L-Lactate Dehydrogenase
  • Malate Dehydrogenase