Cendron Laura, Ramazzina Ileana, Puggioni Vincenzo, Maccacaro Eleonora, Liuzzi Anastasia, Secchi Andrea, Zanotti Giuseppe, Percudani Riccardo
Department of Biomedical Sciences, University of Padova , Padova, Italy.
Department of Life Sciences, University of Parma , Parma, Italy.
Biochemistry. 2016 Nov 22;55(46):6421-6432. doi: 10.1021/acs.biochem.6b00881. Epub 2016 Nov 7.
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 and H 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.
尿囊素的S对映体是几种生物体中嘌呤降解的中间体，也是非类人猿哺乳动物中尿酸分解的最终产物。生物信息学表明，天冬氨酸/谷氨酸消旋酶超家族的蛋白质可能负责最初在假单胞菌属中描述的尿囊素消旋酶（AllR）活性。在这些蛋白质中，催化二元组中的一个半胱氨酸被甘氨酸取代，但重组酶对尿囊素的R和S对映体表现出相似效率（k/K≈5×10 M s）的消旋活性。蛋白质晶体结构确定了位于下游三个残基处的谷氨酸残基（E78），其可在功能上替代缺失的半胱氨酸；通过定点诱变证实了E78的催化作用。尿囊素可通过形成双环中间体（较快）或在手性中心进行质子交换（较慢）而发生消旋。通过用碳和氢核磁共振监测这两种替代机制，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。蛋白质系统发育追踪了作用于谷氨酸的酶中消旋机制的起源，对于谷氨酸这种底物，质子交换是唯一可行的反应机制。尽管催化残基发生了取代，但这种机制通过趋异进化被尿囊素消旋酶继承并得以保留。
