Reaction Mechanism and Catalytic Fingerprint of Allantoin Racemase

Abstract

The stereospecific oxidative decomposition of urate into allantoin is the core of purine catabolism in many organisms. The spontaneous decomposition of upstream intermediates and the nonenzymatic racemization of allantoin lead to an accumulation of (R)-allantoin, because the enzymes converting allantoin into allantoate are specific for the (S) isomer. The enzyme allantoin racemase catalyzes the reversible conversion between the two allantoin enantiomers, thus ensuring the overall efficiency of the catabolic pathway and preventing allantoin accumulation. On the basis of recent crystallographic and biochemical evidence, allantoin racemase has been assigned to the family of cofactor-independent racemases, together with other amino acid racemases. A detailed computational investigation of allantoin racemase has been carried out to complement the available experimental data and to provide atomistic insight into the enzymatic action. Allantoin, the natural substrate of the enzyme, has been investigated at the quantum mechanical level, in order to rationalize its conformational and tautomeric equilibria, playing a key role in protein-ligand recognition and in the following catalytic steps. The reaction mechanism of the enzyme has been elucidated through quantum mechanics/molecular mechanics (QM/MM) calculations. The potential energy surface investigation, carried out at the QM/MM level, revealed a stepwise reaction mechanism. A pair of cysteine residues promotes the stereoinversion of a carbon atom of the ligand without the assistance of cofactors. Electrostatic fingerprint calculations are used to discuss the role of the active site residues in lowering the pK of the substrate. The planar unprotonated intermediate is compared with the enolic allantoin tautomer observed in the active site of the crystallized enzyme. Finally, the enzymatic catalysis featured by allantoin racemase (AllR) is compared with that of other enzymes belonging to the same family.

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“…Previously we have synthesized hydrazide derivatives of o-and n-hydrobenzoic acids (1-4) [27][28][29] which contain thioamide group, ethyl unit and 1,2,4-triazole groups. 1-4 compounds antiradical properties estimation for the purpose of revealing active antioxidants was carried out for the first time.…”

“…Allantoin is known to exist as a racemic mixture of (R) and (S) stereoisomers. A number of studies [27,28] considers the accumulation of (R)-allantoin and (S)-allantoin during the non-enzymatic and enzymatic oxidative urate decomposition into allantoin during the purine catabolism in many organisms. The non-enzymatic racemization of allantoin was found to result in the accumulation of (R)-allantoin, since the enzymes that convert allantoin to allantoate are specific for the (S)-isomer.…”

Biofilm Interaction Mapping and Analysis (BIMA) of Interspecific Interactions in Pseudomonas Co-culture Biofilms

“…Given that PurU is important in maintenance of one carbon pools, the Δ purU mutant may be unable to obtain the reaction products (THF and formate) from N1B4, and as suggested by previous studies in E. coli , may experience glycine starvation due to GlyA inhibition by methionine and adenine ( Nagy et al, 1995 ), both metabolites RCH2 took up from N1B4 based media. In the Δ uraD mutant, which accumulated allantoin, presumably (R)-allantoin, RCH2 would be unable to use this in downstream nitrogen assimilation pathways without a racemase ( Bovigny et al, 2014 ), an enzyme P. stutzeri has been shown to lack ( Van Der Drift et al, 1975 ). Thus UraD is probably essential for converting OHCU into usable (S)-allantoin.…”

Biofilm Interaction Mapping and Analysis (BIMA): A tool for deconstructing interspecific interactions in co-culture biofilms

“…S05). P. stutzeri RCH2 strain JWST9063 (ΔuraD) accumulated allantoin, presumably from spontaneous conversion of 2-oxo-4-hydroxy-4carboxy-5-ureidoimidazoline (OHCU) to (R)-allantoin, which RCH2 would be unable to use in downstream nitrogen assimilation pathways without a racemase (46), an enzyme P. stutzeri has been shown to lack (47). P. stutzeri RCH2 strain JWST9060 (ΔproA) and P. stutzeri RCH2 strain JWST9069 (ΔproB), both known auxotrophs for proline (48), had reduced consumption of glutamate (precursor in proline synthesis) from N1B4, when compared to wild-type (Supplemental Fig.…”