title: Biochemical and mutational studies of the Bacillus cereus CECT 5050T formamidase support the existence of a C-E-E-K tetrad in several members of the nitrilase superfamily.
abstract: Formamidases (EC 3.5.1.49) are poorly characterized proteins. In spite of this scarce knowledge, ammonia has been described as playing a central role in the pathogenesis of human pathogens such as Helicobacter pylori, for which formamidase has been shown to participate in the nitrogen metabolic pathway. Sequence analysis has revealed that at least two different groups of formamidases are classified as EC 3.5.1.49: on the one hand, the derivatives of the FmdA-AmdA superfamily, which are the best studied to date, and on the other hand, the derivatives of Helicobacter pylori AmiF. Here we present the cloning, purification, and characterization of a recombinant formamidase from Bacillus cereus CECT 5050T (BceAmiF), the second member of the AmiF subfamily to be characterized, showing new features of the enzyme further supporting its relationship with aliphatic amidases. We also present homology modeling-based mutational studies confirming the importance of the Glu140 and Tyr191 residues in the enzymatic activities of the AmiF family. Moreover, we can conclude that a second glutamate residue is critical in several members of the nitrilase superfamily, meaning that what has consistently been identified as a C-E-K triad is in fact a C-E-E-K tetrad.
authors.affiliation: Departamento de Química Física, Bioquímica y Química Inorgánica, Universidad de Almería, Almería, Spain.
authors.forename: Pablo
authors.initials: P
authors.lastname: Soriano-Maldonado
authors.forename: Ana Isabel
authors.initials: AI
authors.lastname: Martínez-Gómez
authors.forename: Montserrat
authors.initials: M
authors.lastname: Andújar-Sánchez
authors.forename: José L
authors.initials: JL
authors.lastname: Neira
authors.forename: Josefa María
authors.initials: JM
authors.lastname: Clemente-Jiménez
authors.forename: Francisco Javier
authors.initials: FJ
authors.lastname: Las Heras-Vázquez
authors.forename: Felipe
authors.initials: F
authors.lastname: Rodríguez-Vico
authors.forename: Sergio
authors.initials: S
authors.lastname: Martínez-Rodríguez
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics; D000619:Aminohydrolases / Q000378:metabolism*; D001409:Bacillus cereus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D002384:Catalysis; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism / Q000379:methods; D003001:Cloning, Molecular; D004789:Enzyme Activation; D057075:Enzyme Assays; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D018698:Glutamic Acid / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010802:Phylogeny; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D018698:Glutamic Acid; D000581:Amidohydrolases; C101846:formamidase; D000619:Aminohydrolases; C021867:nitrilase