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_cat/K_M ≈ 5 × 10⁴ M^–1 s^–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 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.

中文翻译：

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微生物尿囊素消旋酶的结构和功能揭示了催化机制的起源和保守性

尿囊素的S对映异构体是几种生物中嘌呤降解的中间体，也是非类人哺乳动物中尿酸溶解的最终产物。生物信息学表明 Asp/Glu 消旋酶超家族的蛋白质可能负责最初在假单胞菌属物种中描述的尿囊素消旋酶 (AllR) 活性。在这些蛋白质中，催化二元组的一个半胱氨酸被甘氨酸取代，但重组酶显示出对R和S具有相似效率的外消旋化活性 ( k _cat / K _M ≈ 5 × 10 ⁴ M ^–1 s ^–1 )尿囊素的对映体。蛋白质晶体结构确定了位于下游三个残基（E78）的谷氨酸残基，可以在功能上替代缺失的半胱氨酸；定点诱变证实了 E78 的催化作用。尿囊素可以通过形成双环中间体（较快）或在手性中心（较慢）进行质子交换而进行消旋化。通过监测¹³ C 和^{1的两种替代机制}H核磁共振，我们发现较快反应的速度不受酶的影响，而较慢反应的速度提高了7个数量级。蛋白质系统发育追溯了作用于谷氨酸的酶的消旋机制的起源，谷氨酸是质子交换是唯一可行的反应机制的底物。这种机制通过不同的进化被尿囊素消旋酶继承，并且尽管催化残基被取代，但仍然保持不变。