In most organisms, malate dehydrogenase (MDH) exists as a homodimeric molecule and is closely related to lactate dehydrogenase (LDH) in structure. It is a large protein molecule with subunits weighing between 30 and 35 kDa. Based on the amino acid sequences, it seems that MDH has diverged into two main phylogenetic groups that closely resemble either mitochondrial isozymes or cytoplasmic/chloroplast isozymes. Because the sequence identity of malate dehydrogenase in the mitochondria is more closely related to its prokaryotic ancestors in comparison to the cytoplasmic isozyme, the theory that mitochondria and chloroplasts were developed through endosymbiosis is plausible. The amino acid sequences of archaeal MDH are more similar to that of LDH than that of MDH of other organisms. This indicates that there is a possible evolutionary linkage between lactate dehydrogenase and malate dehydrogenase.
Each subunit of the malate dehydrogenase dimer has two distinct domains that vary in structure and functionality. A parallel β-sheet structure makes up the NAD+ binding domain, while four β-sheets and one α-helix comprise the central NAD binding site. The subunits are held together through extensive hydrogen-bonding and hydrophobic interactions.
Malate dehydrogenase has also been shown to have a mobile loop region that plays a crucial role in the enzyme's catalytic activity. Studies have shown that conformational change of this loop region from the open conformation to the closed conformation after binding of substrate enhances MDH catalysis through shielding of substrate and catalytic amino acids from solvent. Studies have also indicated that this loop region is highly conserved in malate dehydrogenase.