title: Biochemical and mutational studies of allantoinase from Bacillus licheniformis CECT 20T.
abstract: 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. Co(2+) 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.
authors: Dpto. Química y Física, Universidad de Almería, Campus de Excelencia Internacional Agroalimentario, ceiA3, 04120 Almería, Spain; Centro de Investigación en Biotecnología Agroalimentaria, BITAL, Almería, Spain.
authors: Ana Isabel
authors: AI
authors: Martínez-Gómez
authors: Dpto. Química y Física, Universidad de Almería, Campus de Excelencia Internacional Agroalimentario, ceiA3, 04120 Almería, Spain; Centro de Investigación en Biotecnología Agroalimentaria, BITAL, Almería, Spain.
authors: Pablo
authors: P
authors: Soriano-Maldonado
authors: Dpto. Química y Física, Universidad de Almería, Campus de Excelencia Internacional Agroalimentario, ceiA3, 04120 Almería, Spain; Centro de Investigación en Biotecnología Agroalimentaria, BITAL, Almería, Spain.
authors: Montserrat
authors: M
authors: Andújar-Sánchez
authors: Dpto. Química y Física, Universidad de Almería, Campus de Excelencia Internacional Agroalimentario, ceiA3, 04120 Almería, Spain; Centro de Investigación en Biotecnología Agroalimentaria, BITAL, Almería, Spain.
authors: Josefa María
authors: JM
authors: Clemente-Jiménez
authors: Dpto. Química y Física, Universidad de Almería, Campus de Excelencia Internacional Agroalimentario, ceiA3, 04120 Almería, Spain; Centro de Investigación en Biotecnología Agroalimentaria, BITAL, Almería, Spain.
authors: Felipe
authors: F
authors: Rodríguez-Vico
authors: Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, 03202 Elche, Alicante, Spain; Complex Systems Physics Institute, 50009 Zaragoza, Spain.
authors: José L
authors: JL
authors: Neira
authors: Dpto. Química y Física, Universidad de Almería, Campus de Excelencia Internacional Agroalimentario, ceiA3, 04120 Almería, Spain; Centro de Investigación en Biotecnología Agroalimentaria, BITAL, Almería, Spain.
authors: Francisco Javier
authors: FJ
authors: Las Heras-Vázquez
authors: Dpto. Química y Física, Universidad de Almería, Campus de Excelencia Internacional Agroalimentario, ceiA3, 04120 Almería, Spain; Centro de Investigación en Biotecnología Agroalimentaria, BITAL, Almería, Spain; Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, 03202 Elche, Alicante, Spain; Dpto. Química Física, Universidad de Granada, 18071 Granada, Spain. Electronic address: srodrig@ual.es.
authors: Sergio
authors: S
authors: Martínez-Rodríguez
mesh_terms: D000481:Allantoin / Q000737:chemistry; D000494:Allosteric Regulation; D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D019943:Amino Acid Substitution; D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain; D003035:Cobalt / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000737:chemistry; D006827:Hydantoins / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008345:Manganese / Q000737:chemistry; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D013237:Stereoisomerism; D013379:Substrate Specificity; D044366:Transition Temperature; D014498:Uracil / Q000031:analogs & derivatives / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D006827:Hydantoins; C007419:dihydrouracil; D000481:Allantoin; D003035:Cobalt; D008345:Manganese; D014498:Uracil; D006861:Hydrogen Peroxide; D000581:Amidohydrolases; C019659:allantoinase; D003545:Cysteine
keywords: Allantoinase; Amidohydrolase; TIM-barrel