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 Holorcula marismortui

L-Malate (MalDH) and L-lactate (LDH) dehydrogenases belong to the same fami ly of NAD-dependent enzymes. LDHs are tetramers, whereas MalDHs can be eith er dimeric or tetrameric, To gain insight into molecular relationships betw een LDHs and MalDHs, we studied folding intermediates of a mutant of the LD H-like MalDH (a protein with LDH-like structure and MalDH enzymatic activit y) from the halophilic archaeon Haloarcula marismortui (Hm MalDH). Crystall ographic analysis of Hm MalDH had shown a tetramer made up of two dimers in teracting mainly via complex salt bridge clusters. In the R207S/R292S Hm Ma lDH mutant, these salt bridges are disrupted. Its structural parameters, de termined by neutron scattering and analytical centrifugation under differen t conditions, showed the protein to be a tetramer in 4 M NaCl. At lower sal t concentrations, stable oligomeric intermediates could be trapped at a giv en pH, temperature, or NaCl solvent concentration. The spectroscopic proper ties and enzymatic behavior of monomeric, dimeric, and tetrameric species w ere thus characterized. The properties of the dimeric intermediate were com pared to those of dimeric intermediates of LDH and dimeric MalDHs. A detail ed analysis of the putative dimer-dimer contact regions in these enzymes pr ovided 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.