Biochemical and mutational studies of allantoinase from Bacillus licheniformis CECT 20T

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• 作者：Ana Isabel Mart铆nez-G贸mez ; Pablo Soriano-Maldonado ; Montserrat And煤jar-S谩nchez ; Josefa Mar铆a Clemente-Jim茅nez ; Felipe Rodr铆guez-Vico ; Jos茅 L. Neira ; Francisco Javier Las Heras-V谩zquez ; Sergio Mart铆nez-Rodr铆guez
• 关键词：Allantoinase ; TIM-barrel ; Amidohydrolase
• 刊名：Biochimie
• 出版年：April, 2014
• 年：2014
• 卷：99
• 期：Complete
• 页码：178-188
• 全文大小：1058 K

文摘

Allantoinases (allantoin amidohydrolase, E.C. 3.5.2.5) catalyze the hydrolysis of the amide bond of allantoin to form allantoic acid, in those organisms where allantoin is not the final product of uric acid degradation. Despite their importance in the purine catabolic pathway, sequences of microbial allantoinases with proven activity are scarce, and only the enzyme from Escherichia coli (AllEco) has been studied in detail in the genomic era. In this work, we report the cloning, purification and characterization of the recombinant allantoinase from Bacillus licheniformis CECT 20T (AllBali). The enzyme was a homotetramer with an apparent T_m of 62聽卤聽1聽掳C. Optimal parameters for the enzyme activity were pH 7.5 and 50聽掳C, showing apparent K_m and k_cat values of 17.7聽卤聽2.7聽mM and 24.4聽卤聽1.5聽s^鈭?, respectively. Co²⁺ proved to be the most effective cofactor, inverting the enantioselectivity of AllBali when compared to that previously reported for other allantoinases. The common ability of different cyclic amidohydrolases to hydrolyze distinct substrates to the natural one also proved true for AllBali. The enzyme was able to hydrolyze hydantoin, dihydrouracil and 5-ethyl-hydantoin, although at relative rates 3-4 orders of magnitude lower than with allantoin. Mutagenesis experiments suggest that S292 is likely implicated in the binding of the allantoin ring through the carbonyl group of the polypeptide main chain, which is the common mechanism observed in other members of the amidohydrolase family. In addition, our results suggest an allosteric effect of H₂O₂ toward allantoinase.