Biochemical properties of a Pseudomonas aminotransferase involved in caprolactam metabolism

Abstract

The biodegradation of the nylon‐6 precursor caprolactam by a strain of Pseudomonas jessenii proceeds via ATP‐dependent hydrolytic ring opening to 6‐aminohexanoate. This non‐natural ω‐amino acid is converted to 6‐oxohexanoic acid by an aminotransferase (PjAT) belonging to the fold type I pyridoxal 5′‐phosphate (PLP) enzymes. To understand the structural basis of 6‐aminohexanoatate conversion, we solved different crystal structures and determined the substrate scope with a range of aliphatic and aromatic amines. Comparison with the homologous aminotransferases from Chromobacterium violaceum (CvAT) and Vibrio fluvialis (VfAT) showed that the PjAT enzyme has the lowest KM values (highest affinity) and highest specificity constant (kcat/KM) with the caprolactam degradation intermediates 6‐aminohexanoate and 6‐oxohexanoic acid, in accordance with its proposed in vivo function. Five distinct three‐dimensional structures of PjAT were solved by protein crystallography. The structure of the aldimine intermediate formed from 6‐aminohexanoate and the PLP cofactor revealed the presence of a narrow hydrophobic substrate‐binding tunnel leading to the cofactor and covered by a flexible arginine, which explains the high activity and selectivity of the PjAT with 6‐aminohexanoate. The results suggest that the degradation pathway for caprolactam has recruited an aminotransferase that is well adapted to 6‐aminohexanoate degradation. Database The atomic coordinates and structure factors P. jessenii 6‐aminohexanoate aminotransferase have been deposited in the PDB as entries http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6G4B (E∙succinate complex), http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6G4C (E∙phosphate complex), http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6G4D (E∙PLP complex), http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6G4E (E∙PLP‐6‐aminohexanoate intermediate), and http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6G4F (E∙PMP complex).

The crystal structure of the tetrameric DABA‐aminotransferase EctB, a rate‐limiting enzyme in the ectoine biosynthesis pathway

“…Class III x-TAs, such as the herein presented EctB, must have dual substrate recognition mechanisms to differentiate between two substrate pairs [39,40] and be able to specifically catalyze the transfer of the distal amine group. Several studies show that the active site of many aminotransferases has two binding pockets, denoted according to their relative position to the PLP coenzyme (Figs 1C and 2), and that the orientation of substrates in the active site is often coordinated by one or two arginine residues that form strong directional bonds to the substrate's carboxylate group [39,[41][42][43][44][45][46] by a so-called end-on geometry [47]. When the arginine-carboxylate bond is not required, some aminotransferases neutralize the arginine through a strong ionic bond with a glutamate residue (i.e., glutamate switch), while others have a flexible arginine that moves in and out of the active site (i.e., arginine switch; Fig.…”

Robust ω-Transaminases by Computational Stabilization of the Subunit Interface

“…The vector pET-20b (+)-His- Pj TA, containing an in-frame fusion of the Pj TA gene with the ATG start codon, was described in our previous work. 12 Mutations of Pj TA were created by QuikChange site-directed mutagenesis. Primers were designed by the QuikChange Primer Design Program of Agilent Technologies.…”

“…Pj TA is a homodimeric PLP fold-type I enzyme with subunits of 456 amino acids. 12 The enzyme catalyzes the deamination of 6-aminohexanoate, which is the first intermediate in the bacterial degradation of the industrial nylon precursor caprolactam. The structures of the apoenzyme, external aldimine with 6-aminohexanoate, and PMP-bound enzyme were solved by protein crystallography.…”

“…The structures of the apoenzyme, external aldimine with 6-aminohexanoate, and PMP-bound enzyme were solved by protein crystallography. 12 …”

Asymmetric Synthesis of Optically Pure Aliphatic Amines with an Engineered Robust ω-Transaminase

“…The discovery [22] and characterization [23] of a class III (S)-selective ω-TA from Pseudomonas jessenii (PjTA) were described previously. Identified as a key enzyme in the caprolactam degradation pathway of P. jessenii, PjTA converts 6-aminohexanoic acid (6-AHA) to 6-oxohexanoic acid (6-OHA).…”