Abstract
ABSTRACTWe previously isolated the transaminase KES23458 from Pseudomonas sp. strain AAC as a promising biocatalyst for the production of 12-aminododecanoic acid, a constituent building block of nylon-12. Here, we report the subsequent characterization of this transaminase. It exhibits activity with a broad substrate range which includes α-, β-, and ω-amino acids, as well as α,ω-diamines and a number of other industrially relevant compounds. It is therefore a prospective candidate for the biosynthesis of a range of polyamide monomers. The crystal structure of KES23458 revealed that the protein forms a dimer containing a large active site pocket and unusual phosphorylated histidine residues. To infer the physiological role of the transaminase, we expressed, purified, and characterized a dehydrogenase from the same operon, KES23460. Unlike the transaminase, the dehydrogenase was shown to be quite selective, catalyzing the oxidation of malonic acid semialdehyde, formed from β-alanine transamination via KES23458. In keeping with previous reports, the dehydrogenase was shown to catalyze both a coenzyme A (CoA)-dependent reaction to form acetyl-CoA and a significantly slower CoA-independent reaction to form acetate. These findings support the original functional assignment of KES23458 as a β-alanine transaminase. However, a seemingly well-adapted active site and promiscuity toward unnatural compounds, such as 12-aminododecanoic acid, suggest that this enzyme could perform multiple functions for Pseudomonas sp. strain AAC.IMPORTANCE We describe the characterization of an industrially relevant transaminase able to metabolize 12-aminododecanoic acid, a constituent building block of the widely used polymer nylon-12, and we report the biochemical and structural characterization of the transaminase protein. A physiological role for this highly promiscuous enzyme is proposed based on the characterization of a related gene from the host organism. Molecular dynamics simulations were carried out to compare the conformational changes in the transaminase protein to better understand the determinants of specificity in the protein. This study makes a substantial contribution that is of interest to the broad biotechnology and enzymology communities, providing insights into the catalytic activity of an industrially relevant biocatalyst as well as the biological function of this operon.
Highlights
We previously isolated the transaminase KES23458 from Pseudomonas sp. strain AAC as a promising biocatalyst for the production of 12-aminododecanoic acid, a constituent building block of nylon-12
It is perhaps underappreciated that enzymes can possess very broad substrate specificities, a key requirement for their evolutionary adaptability, as this allows for the development of new catalytic capabilities when required for survival or competitive advantage [1,2,3]
The ability of microorganisms to degrade nonnatural chemicals, such as pesticides or synthetic antibiotics, has resulted in new metabolic pathways for resistance and catabolism over relatively short periods of evolutionary time [4, 5]. These proteins often evolve for catabolic purposes in the microorganisms involved, these newly evolved functions represent an opportunity from a biocatalytic perspective
Summary
We previously isolated the transaminase KES23458 from Pseudomonas sp. strain AAC as a promising biocatalyst for the production of 12-aminododecanoic acid, a constituent building block of nylon-12. We report the subsequent characterization of this transaminase It exhibits activity with a broad substrate range which includes ␣-, -, and -amino acids, as well as ␣,-diamines and a number of other industrially relevant compounds. In keeping with previous reports, the dehydrogenase was shown to catalyze both a coenzyme A (CoA)-dependent reaction to form acetyl-CoA and a significantly slower CoA-independent reaction to form acetate These findings support the original functional assignment of KES23458 as a -alanine transaminase. -Transaminases (-TAs) are renowned for their promiscuity and have exhibited catalytic prowess in the synthesis of unnatural compounds [6] The ability of these enzymes to act on foreign molecules has drawn widespread interest from both academia and industry, and while they are used widely in the synthesis of chiral building blocks [7], they can be used to produce more complex synthetic pharmaceuticals. Given the low homology (Ͻ30%) among the class [14, 15], an understanding of the sequence-function relationship of these enzymes can be challenging and is typically only possible with a detailed understanding of the interactions between an enzyme and its substrate [8, 16]
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