DCoH and DCoHalpha are bifunctional proteins that function as 4a-hydroxytetrahydrobiopterin dehydratases and as coactivators of HNF1alpha-dependent transcription. Although these isoforms share sequence and structural similarity and equivalent enzyme activities, DCoH is a hyperstable tetramer whereas DCoHalpha readily forms dimers. Differences in quaternary structure affect the formation of the DCoH(alpha):HNF1alpha complex. Because the interface used to bind HNF1alpha is masked in tetrameric DCoH, the DCoH:HNF1alpha complex is only formed in vivo, presumably by co-translational folding. Conversely, the DCoHalpha:HNF1alpha complex readily forms in vitro. We identified residues in DCoHalpha that differed from those in the dimer-dimer interface of tetrameric DCoH. Mutating these residues altered the quaternary state and concomitantly the ability of the mutated proteins to affect HNF1alpha-dependent DNA binding. Our results indicate that three residues, Asn61, Gln45, and Lys98 in DCoHalpha play a role in oligomeric flexibility, which enables DCoHalpha to more readily interact with HNF1alpha and increase DNA binding.