Revisiting D‐Acylases for D‐Amino Acid Production
ABSTRACT 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 t...
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Wiley
2025-06-01
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| Series: | Microbial Biotechnology |
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| Online Access: | https://doi.org/10.1111/1751-7915.70179 |
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| author | Sergio Martínez‐Rodríguez Jose Antonio Gavira |
| author_facet | Sergio Martínez‐Rodríguez Jose Antonio Gavira |
| author_sort | Sergio Martínez‐Rodríguez |
| collection | DOAJ |
| description | ABSTRACT 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. |
| format | Article |
| id | doaj-art-fffa7069248d4422ac1af86ab79f4d67 |
| institution | Kabale University |
| issn | 1751-7915 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | Wiley |
| record_format | Article |
| series | Microbial Biotechnology |
| spelling | doaj-art-fffa7069248d4422ac1af86ab79f4d672025-08-20T03:30:02ZengWileyMicrobial Biotechnology1751-79152025-06-01186n/an/a10.1111/1751-7915.70179Revisiting D‐Acylases for D‐Amino Acid ProductionSergio Martínez‐Rodríguez0Jose Antonio Gavira1Department of Biochemistry and Molecular Biology III and Immunology University of Granada Granada SpainRaw Materials, Human and Environmental Health, UGR Associated Unit of the CSIC by the IACT‐CSIC Armilla Granada SpainABSTRACT 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.https://doi.org/10.1111/1751-7915.70179acylaseamidohydrolaseamidohydrolase processamino acidenzyme cascadeN‐acyl‐amino acid |
| spellingShingle | Sergio Martínez‐Rodríguez Jose Antonio Gavira Revisiting D‐Acylases for D‐Amino Acid Production Microbial Biotechnology acylase amidohydrolase amidohydrolase process amino acid enzyme cascade N‐acyl‐amino acid |
| title | Revisiting D‐Acylases for D‐Amino Acid Production |
| title_full | Revisiting D‐Acylases for D‐Amino Acid Production |
| title_fullStr | Revisiting D‐Acylases for D‐Amino Acid Production |
| title_full_unstemmed | Revisiting D‐Acylases for D‐Amino Acid Production |
| title_short | Revisiting D‐Acylases for D‐Amino Acid Production |
| title_sort | revisiting d acylases for d amino acid production |
| topic | acylase amidohydrolase amidohydrolase process amino acid enzyme cascade N‐acyl‐amino acid |
| url | https://doi.org/10.1111/1751-7915.70179 |
| work_keys_str_mv | AT sergiomartinezrodriguez revisitingdacylasesfordaminoacidproduction AT joseantoniogavira revisitingdacylasesfordaminoacidproduction |