Structure and function of a microbial allantoin racemase reveal the origin and conservation of a catalytic mechanism

The S enantiomer of allantoin is an intermediate of purine degradation in several organisms and the final product of uricolysis in non-hominoid mammals. Bioinformatics indicated that proteins of the Asp/Glu racemase superfamily could be responsible for the allantoin racemase (AllR) activity originally described in Pseudomonas species. In these proteins, a cysteine of the catalytic dyad is substituted by glycine, yet the recombinant enzyme displayed racemization activity with similar efficiency (kcat/Km ≈ 5*10(4) s(-1) M(-1)) for the R and S enantiomers of allantoin. The protein crystal structure identified a glutamate residue located three residues downstream (E78) that can functionally replace the missing cysteine; the catalytic role of E78 was confirmed by site-directed mutagenesis. Allantoin can undergo racemization through formation of a bicyclic intermediate (faster) or proton exchange at the chiral center (slower). By monitoring the two alternative mechanisms by 13C and 1H NMR, we found that the velocity of the faster reaction is unaffected by the enzyme, whereas the velocity of the slower reaction is increased by seven orders of magnitude. Protein phylogenies trace the origin of the racemization mechanism in enzymes acting on glutamate, a substrate for which proton exchange is the only viable reaction mechanism. This mechanism was inherited by allantoin racemase through divergent evolution, and conserved in spite of the substitution of catalytic residues