文摘

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 gaininsight into molecular relationships between LDHs and MalDHs, we studied folding intermediates of amutant of the LDH-like MalDH (a protein with LDH-like structure and MalDH enzymatic activity) fromthe halophilic archaeon Haloarcula marismortui (Hm MalDH). Crystallographic analysis of Hm MalDHhad shown a tetramer made up of two dimers interacting mainly via complex salt bridge clusters. In theR207S/R292S Hm MalDH mutant, these salt bridges are disrupted. Its structural parameters, determinedby neutron scattering and analytical centrifugation under different conditions, showed the protein to be atetramer in 4 M NaCl. At lower salt concentrations, stable oligomeric intermediates could be trapped ata given pH, temperature, or NaCl solvent concentration. The spectroscopic properties and enzymaticbehavior of monomeric, dimeric, and tetrameric species were thus characterized. The properties of thedimeric intermediate were compared to those of dimeric intermediates of LDH and dimeric MalDHs. Adetailed analysis of the putative dimer-dimer contact regions in these enzymes provided an explanationof why some can form tetramers and others cannot. The study presented here makes Hm MalDH the bestcharacterized example so far of an LDH-like MalDH.