Biochemical and mutational studies of allantoinase from Bacillus licheniformis CECT 20T
| Autor: | Ana Isabel Martínez-Gómez, Francisco Javier Las Heras-Vázquez, Montserrat Andújar-Sánchez, José L. Neira, Sergio Martínez-Rodríguez, Josefa María Clemente-Jiménez, Felipe Rodríguez-Vico, Pablo Soriano-Maldonado |
|---|---|
| Rok vydání: | 2014 |
| Předmět: |
Allantoic acid
Stereochemistry Allantoinase Hydantoin Bacillus Biochemistry Amidohydrolases Substrate Specificity chemistry.chemical_compound Allantoin Allosteric Regulation Bacterial Proteins Catalytic Domain Transition Temperature Cysteine Bacillus licheniformis Enzyme kinetics Enzyme Inhibitors Uracil Manganese biology Amidohydrolase Hydantoins Hydrolysis Stereoisomerism Cobalt Hydrogen Peroxide General Medicine Hydrogen-Ion Concentration biology.organism_classification Molecular Weight Kinetics Amino Acid Substitution chemistry Mutagenesis Site-Directed Protein Binding Homotetramer |
| Zdroj: | Biochimie. 99:178-188 |
| ISSN: | 0300-9084 |
| DOI: | 10.1016/j.biochi.2013.12.002 |
| Popis: | 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 Tm of 62 ± 1 °C. Optimal parameters for the enzyme activity were pH 7.5 and 50 °C, showing apparent Km and kcat values of 17.7 ± 2.7 mM and 24.4 ± 1.5 s−1, 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. |
| Databáze: | OpenAIRE |
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