Introduction of Asymmetry in the Fused 4-Oxalocrotonate Tautomerases

Members of the 4-oxalocrotonate tautomerase (4OT) subgroup in the tautomerase superfamily (TSF) are constructed from a single beta-alpha-beta unit and form homo- or heterohexamers, whereas those of the other four subgroups are composed of two consecutively joined beta-alpha-beta units and form trimers. A subset of sequences, double the length of the short 4OTs, is found in the 4-OT subgroup. These "fused" 4-OTs form a separate subgroup that connects to the short 4-OTs in a sequence similarity network (SSN). The fused gene can be a template for the other four subgroups, resulting in the diversification of activity. Analysis of the SSN shows that multiple nodes in the fused 4-OTs connect to five linker nodes, which in turn connect to the short 4OTs. Some fused 4-OTs are symmetric trimers and others are asymmetric trimers. The origin of this asymmetry was investigated by subjecting the sequences in three linker nodes and a closely associated fourth node to kinetic, mutagenic, and structural analyses. The results show that each sequence corresponds to the alpha- or beta-subunit of a heterohexamer that functions as a 4-OT. Mutagenesis indicates that the key residues in both are alpha Pro1 and beta Arg-11, like that of a typical 4-OT. Crystallographic analysis shows that both heterohexamers are asymmetric, where one heterodimer is flipped 180 degrees relative to the other two heterodimers. The fusion of two subunits (alpha and beta) of one asymmetric heterohexamer generates an asymmetric trimer with 4-OT activity. Hence, asymmetry can be introduced at the heterohexamer level and then retained in the fused trimers.