Transaminases are attractive catalysts for the production of enantiopure amines. However, the poor stability of these enzymes often limits their application in biocatalysis. Here, we used a framework for enzyme stability engineering by computational library design (FRESCO) to stabilize the homodimeric PLP fold type I ω-transaminase from Pseudomonas jessenii . A large number of surface-located point mutations and mutations predicted to stabilize the subunit interface were examined. Experimental screening revealed that 10 surface mutations out of 172 tested were indeed stabilizing (6% success), whereas testing 34 interface mutations gave 19 hits (56% success). Both the extent of stabilization and the spatial distribution of stabilizing mutations showed that the subunit interface was critical for stability. After mutations were combined, 2 very stable variants with 4 and 6 mutations were obtained, which in comparison to wild type ( T m app = 62 °C) displayed T m app values of 80 and 85 °C, respectively. These two variants were also 5-fold more active at their optimum temperatures and tolerated high concentrations of isopropylamine and cosolvents. This allowed conversion of 100 mM acetophenone to ( S )-1-phenylethylamine (>99% enantiomeric excess) with high yield (92%, in comparison to 24% with the wild-type transaminase). Crystal structures mostly confirmed the expected structural changes and revealed that the most stabilizing mutation, I154V, featured a rarely described stabilization mechanism: namely, removal of steric strain. The results show that computational interface redesign can be a rapid and powerful strategy for transaminase stabilization.

Some bacterial cultures are capable of growth on caprolactam as sole carbon and nitrogen source, but the enzymes of the catabolic pathway have not been described. We isolated a caprolactam-degrading strain of Pseudomonas jessenii from soil and identified proteins and genes putatively involved in caprolactam metabolism using quantitative mass spectrometry-based proteomics. This led to the discovery of a caprolactamase and an aminotransferase that are involved in the initial steps of caprolactam conversion. Additionally, various proteins were identified that likely are involved in later steps of the pathway. The caprolactamase consists of two subunits and demonstrated high sequence identity to the 5-oxoprolinases. Escherichia coli cells expressing this caprolactamase did not convert 5-oxoproline but were able to hydrolyze caprolactam to form 6-aminocaproic acid in an ATP-dependent manner. Characterization of the aminotransferase revealed that the enzyme deaminates 6-aminocaproic acid to produce 6-oxohexanoate with pyruvate as amino acceptor. The amino acid sequence of the aminotransferase showed high similarity to subgroup II ω-aminotransferases of the PLP-fold type I proteins. Finally, analyses of the genome sequence revealed the presence of a caprolactam catabolism gene cluster comprising a set of genes involved in the conversion of caprolactam to adipate.Electronic supplementary materialThe online version of this article (10.1007/s00253-018-9073-7) contains supplementary material, which is available to authorized users.

We constructed an enzymatic network composed of three different enzymes for the synthesis of valuable ether amines. The enzymatic reactions are interconnected to catalyze the oxidation and subsequent transamination of the substrate and to provide cofactor recycling. This allows production of the desired ether amines from the corresponding ether alcohols with inorganic ammonium as the only additional substrate. To examine conversion, individual and overall reaction equilibria were established. Using these data, it was found that the experimentally observed conversions of up to 60% observed for reactions containing 10 mM alcohol and up to 280 mM ammonia corresponded well to predicted conversions. The results indicate that efficient amination can be driven by high concentrations of ammonia and may require improving enzyme robustness for scale-up. Biotechnol. Bioeng. 2016;113: 1853-1861. © 2016 Wiley Periodicals, Inc.

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).

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