The Structure and Function of a Microbial Allantoin Racemase Reveal the Origin and Conservation of a Catalytic Mechanism

Abstract

The S enantiomer of allantoin is an intermediate of purine degradation in several organisms and the final product of uricolysis in nonhominoid 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 with glycine, yet the recombinant enzyme displayed racemization activity with a similar efficiency (k/K ≈ 5 × 10 M s) 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 C andH nuclear magnetic resonance, we found that the velocity of the faster reaction is unaffected by the enzyme, whereas the velocity of the slower reaction is increased by 7 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.

Unveiling an indole alkaloid diketopiperazine biosynthetic pathway that features a unique stereoisomerase and multifunctional methyltransferase

“…Phylogenetic comparison of the NozR sequence with sequences of functionally characterized homologs supported NozR as a member of the widespread Asp/Glu racemase family (Fig. 4 ) 33 – 38 . Prototypical family members catalyze bidirectional racemization with an acid-base catalytic dyad, commonly of cysteine residues.…”

Through the Looking Glass: Chiral Recognition of Substrates and Products at the Active Sites of Racemases and Epimerases

“…[179,180] Consequently,A llanR (EC 5.1.99.3) plays the vital role of converting (R)-allanotin formed through nonenzymatic processes to (S)-allantoin for further catabolism. [181,182] This cofactor-independent racemase utilizes at wobase mechanismwith either Cys184 and Cys79 (Klebsiella pneumoniae enzyme) [183] or Glu180 and Cys78 (Pseudomonasfluorescens enzyme) [184] acting as the enantiospecific Brønsted bases to abstract the a-proton from (R)-and (S)-allantoin, respectively,w ith the oxygen of the enolate intermediate being stabilized by an oxyanion hole (Scheme10).…”

Immobilization of Allantoinase for the Development of an Optical Biosensor of Oxidative Stress States

“…CD kinetics were collected following the decreasing signal at 210 nm, corresponding to (S)-allantoin consumption. Racemization rate of the residual (R)-allantoin was considered negligible in the time windows required for the kinetics measurements [47,48]. Moreover, the reverse stereoselective condensation of allantoate to form (S)-allantoin does not affect the kinetic signal, because of the lesser puuE efficiency in catalyzing this reverse reaction [23].…”