Revisiting D-Acylases for D-Amino Acid Production
N-Acyl-D-amino acid deacylases (EC 3.5.1.81, also known as D-acylases) have been studied for decades for their utility in the kinetic resolution of N-acetyl-D,L-amino acids (NAAs) due to a marked stereospecificity. In conjunction with an N-succinyl-amino acid racemase (NSAR), they impulse the dynami...
| Autores: | , |
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| Tipo de recurso: | artículo |
| Estado: | Versión publicada |
| Fecha de publicación: | 2025 |
| País: | España |
| Institución: | Consejo Superior de Investigaciones Científicas (CSIC) |
| Repositorio: | DIGITAL.CSIC. Repositorio Institucional del CSIC |
| OAI Identifier: | oai:digital.csic.es:10261/396095 |
| Acceso en línea: | http://hdl.handle.net/10261/396095 https://doi.org/10.1111/1751-7915.70179 |
| Access Level: | acceso abierto |
| Palabra clave: | Acylase Amidohydrolase Amidohydrolase process Amino acid Enzyme cascade N-acyl-amino acid N-acyl-D-amino acid deacylase N-succinyl-amino acid racemase Racemase |
| Sumario: | N-Acyl-D-amino acid deacylases (EC 3.5.1.81, also known as D-acylases) have been studied for decades for their utility in the kinetic resolution of N-acetyl-D,L-amino acids (NAAs) due to a marked stereospecificity. In conjunction with an N-succinyl-amino acid racemase (NSAR), they impulse the dynamic kinetic resolution (DKR) of different NAAs until the corresponding enantiomerically pure D-amino acids. Besides the clear interest in this enzyme cascade, the application of D-acylase/NSAR tandems has been only briefly described outside the industrial field. In this work, we revisit D-acylases for the DKR of NAAs, reporting the characterisation of two new recombinant D-acylases belonging to Bordetella petrii and Klebsiella pneumoniae. The enzymes were successfully coupled with the recombinant NSAR from Geobacillus stearothermophilus for the biosynthesis of D-methionine or D-aminobutyric acid. We also carried out the structural characterisation of the D-acylase from Klebsiella pneumoniae (KleDacyl), providing the second experimental 3-D structure of a member of this family of enzymes. The structural model shows a highly dynamic character of this amidohydrolase superfamily member, supplying a snapshot of an open conformation of the enzyme most likely preceding substrate entrance into the catalytic cleft. Our results confirm for the first time the importance of an α/β mobile domain in the substrate specificity of D-acylases (region 282–341 in KleDacyl), opening up new strategies for structural-based protein engineering strategies. |
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