title: Biochemical and Mutational Characterization of N-Succinyl-Amino Acid Racemase from Geobacillus stearothermophilus CECT49.
abstract: N-Succinyl-amino acid racemase (NSAAR), long referred to as N-acyl- or N-acetyl-amino acid racemase, is an enolase superfamily member whose biotechnological potential was discovered decades ago, due to its use in the industrial dynamic kinetic resolution methodology first known as "Acylase Process". In previous works, an extended and enhanced substrate spectrum of the NSAAR from Geobacillus kaustophilus CECT4264 toward different N-substituted amino acids was reported. In this work, we describe the cloning, purification, and characterization of the NSAAR from Geobacillus stearothermophilus CECT49 (GstNSAAR). The enzyme has been extensively characterized, showing a higher preference toward N-formyl-amino acids than to N-acetyl-amino acids, thus confirming that the use of the former substrates is more appropriate for a biotechnological application of the enzyme. The enzyme showed an apparent thermal denaturation midpoint of 77.0 ± 0.1 °C and an apparent molecular mass of 184 ± 5 kDa, suggesting a tetrameric species. Optimal parameters for the enzyme activity were pH 8.0 and 55-65 °C, with Co(2+) as the most effective cofactor. Mutagenesis and binding experiments confirmed K166, D191, E216, D241, and K265 as key residues in the activity of GstNSAAR, but not indispensable for substrate binding.
authors: Dpto. Química y Física, Universidad de Almería, Campus de Excelencia Internacional Agroalimentario, ceiA3, Edificio CITE I, Carretera de Sacramento s/n., 04120, La Cañada de San Urbano, Almería, Spain.
authors: Pablo
authors: P
authors: Soriano-Maldonado
authors: Montserrat
authors: M
authors: Andújar-Sánchez
authors: Josefa María
authors: JM
authors: Clemente-Jiménez
authors: Felipe
authors: F
authors: Rodríguez-Vico
authors: Francisco Javier
authors: FJ
authors: Las Heras-Vázquez
authors: Sergio
authors: S
authors: Martínez-Rodríguez
mesh_terms: D000591:Amino Acid Isomerases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020134:Catalytic Domain; D003001:Cloning, Molecular; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D016296:Mutagenesis; D011489:Protein Denaturation; D055503:Protein Multimerization
chemical_list: D001426:Bacterial Proteins; D000591:Amino Acid Isomerases