Abstract

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 Escherichia coli, isolated by His‐tag affinity chromatography, and subjected to functional and structural studies. Activity toward caprolactam required ATP and was dependent on the presence of bicarbonate in the assay buffer. The hydrolysis product was identified as 6‐aminocaproic acid. 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.

Highlights

  • Whereas plasmids encoding caprolactam degradation genes were discovered in Pseudomonas putida,[10,11] the enzymes involved were not characterized until we recently described the pathway in the caprolactam-utilizing bacterium P. jessenii strain GO3.6 Proteomics analysis indicated that cells growing on caprolactam produced large amounts of two different polypeptides with sequence similarity to eukaryotic oxoprolinases

  • To comply with the terminology proposed by Weidenweber et al[15] for acetone carboxylase we call the expected ATP-binding subunit of caprolactamase subunit α or CapA and the other subunit β or CapB; these align to the oxoprolinase sequence in the order of A-B, but are encoded on the P. jessenii genome and in the expression clone in the order of B-A

  • Caprolactamase belongs to a group of ATP-dependent hydrolases of the oxoprolinase/ hydantoinase family of enzymes

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Summary

| INTRODUCTION

Ε-Caprolactam is used as the building block for the homopolymer nylon-6. Non-reacted caprolactam may be discharged via wastewater at nylon-6 production plants. Carboxylase.[6,17,18] Like caprolactamase, hydantoinase and 5-oxoprolinase catalyze ATP-dependent hydrolytic lactam ring opening (Figure 1A-C).[12,13] CapAB has no 5-oxoprolinase activity and to our knowledge it is the only enzyme of this group that is shown to convert caprolactam. It has been proposed that hydrolysis of 5-oxoproline proceeds via phosphorylation of the iminol tautomer in the large subunit and lactam cleavage in the other subunit.[25] Crystal structures of acetone carboxylase from Xanthobacter autotrophicus (XaAc, PDB) and acetophenone carboxylase from Aromatoleum aromaticum (AaApc, PDB) were recently published[14,15] and suggested that different subunits are involved in ATP-dependent substrate phosphorylation and carbon-carbon bond formation. The activities of the mutants and observation that caprolactam hydrolysis was dependent on the presence of bicarbonate suggested possible mechanisms for ATP-dependent lactam hydrolysis

| RESULTS AND DISCUSSION
86 Q88H51
| CONCLUSIONS
| MATERIALS AND METHODS

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