Catalytic and structural properties of ATP ‐dependent caprolactamase from Pseudomonas jessenii
| Autor: | Hein J. Wijma, Henriëtte J. Rozeboom, Meintje S de Vries, Dick B. Janssen, Antonija Marjanovic, Clemens Mayer, Marleen Otzen |
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| Přispěvatelé: | Biotechnology, Stratingh Institute of Chemistry, Biomolecular Chemistry & Catalysis |
| Rok vydání: | 2021 |
| Předmět: |
Models
Molecular Protein Conformation alpha-Helical Lactamase Gene Expression Crystallography X-Ray Biochemistry Substrate Specificity chemistry.chemical_compound Adenosine Triphosphate Structural Biology Caprolactam Cloning Molecular Research Articles chemistry.chemical_classification 0303 health sciences 5‐oxoproline 6‐aminocaproic acid biology phosphorylation Chemistry Hydrolysis 030302 biochemistry & molecular biology Recombinant Proteins 5-oxoproline Aminocaproic Acid Lactam Thermodynamics Research Article Protein Binding Stereochemistry Genetic Vectors Pseudomonas jessenii Amidohydrolases Structure-Activity Relationship 03 medical and health sciences nylon 6 Bacterial Proteins Affinity chromatography Tetramer Pseudomonas Hydrolase Escherichia coli Protein Interaction Domains and Motifs Amino Acid Sequence carboxylase Molecular Biology 030304 developmental biology Binding Sites Sequence Homology Amino Acid biology.organism_classification Protein Subunits Enzyme Mutation Protein Conformation beta-Strand Protein Multimerization hydrolase Sequence Alignment 6-aminocaproic acid |
| Zdroj: | Proteins Proteins, 89(9):26082, 1079-1098. Wiley |
| ISSN: | 1097-0134 0887-3585 |
| Popis: | Caprolactamase is the first enzyme in the caprolactam degradation pathway of Pseudomonas jessenii. It is composed of two subunits (CapA and CapB) and sequence-related to other ATP-dependent enzymes involved in lactam hydrolysis, like 5-oxoprolinases and hydantoinases. Low sequence similarity also exists with ATP-dependent acetone- and acetophenone carboxylases. The caprolactamase was produced in E. coli, isolated by His-tag affinity chromatography, and subjected to functional and structural studies. Activity towards caprolactam required ATP and was dependent on the presence of bicarbonate in the assay buffer. The hydrolysis product was identified as 6-aminocaproic acid (6-ACA). Quantum mechanical modeling indicated that the hydrolysis of caprolactam was highly disfavored (ΔG0 ' = 23 kJ/mol), which explained the ATP dependence. A crystal structure showed that the enzyme exists as an (αβ)2 tetramer and revealed an ATP-binding site in CapA and a Zn-coordinating site in CapB. Mutations in the ATP-binding site of CapA (D11A and D295A) significantly reduced product formation. Mutants with substitutions in the metal binding site of CapB (D41A, H99A, D101A and H124A) were inactive and less thermostable than the wild-type enzyme. These residues proved to be essential for activity and on basis of the experimental findings we propose possible mechanisms for ATP-dependent lactam hydrolysis. This article is protected by copyright. All rights reserved. |
| Databáze: | OpenAIRE |
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