Structural insights into the substrate specificity of 5-chloro-2-hydroxymuconate tautomerase CnbG.

The catechol meta-cleavage pathway is widely involved in the degradation of aromatic compounds, including those halogenated aromatic hydrocarbons and their derivatives. CnbG is a kind of 4-oxalocrotonate tautomerase (4-OT) located in the catechol meta-cleavage pathway, catalyzes the ketonization of cis,cis-5-chloro-2-hydroxymuconate and cis,cis-2-hydroxymuconate to yield 5-chloro-2-oxo-3-hexene-1,6-dioate and 2-oxo-3-hexene-1,6-dioate, and contributes to the degradation of 4-chloronitrobenzene and chlorobenzene in Comamonas testosteroni CNB-1. Yet, the reason why CnbG and those 4-OTs could recognize various substrates is not well explained. Here, we determined the crystal structure of CnbG at resolution of 2.0 Å and identified that the potential substrate pocket involved in four conserved residues, residues Pro1, Arg11, Arg39 and Trp50, but not five conserved residues as those reported in other 4-OTs. We also found the four conserved residues assemble different sequence patterns in different 4-OTs, indicating their potential roles in catalysis and substrate binding. Via molecular docking, we found the 5-chloro group was clamped by two residues and extended to the solvent, indicating a substrate binding mode that could bear the substitution of different groups in the 5-position. Our work extends the knowledge of the substrate specificity of enzymes in the catechol meta-cleavage pathway.