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Abstract

We previously isolated the transaminase 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 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, . Unlike the transaminase, the dehydrogenase was shown to be quite selective, catalyzing the oxidation of malonic acid semialdehyde, formed from β-alanine transamination via . 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 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.

INTRODUCTION

Previously, we characterized a number of transaminases from Pseudomonas sp. strain AAC (NCBI accession number ), some of which were able to metabolize 12-aminododecanoic acid, a nonnatural compound with potential as a building block for industrial nylon-12 synthesis (). Hazards associated with the synthesis of nylon-12 (), as well as global shortages of the material in recent years (), have afforded opportunities for the application of biocatalysis within the materials field (), and a number of examples have been reported to date (, ). However, while most organisms are unable to metabolize 12-aminododecanoic acid, the Pseudomonas sp. strain AAC bacterium contains three enzymes with activity against the compound. Catalytic promiscuity is often thought to be the result of an enzymatic activity that was acquired coincidentally and evolved under selective pressure. In this case, the substrate is neither toxic nor abundant in the environment, and it is unlikely that there was a strong selective pressure to evolve with a metabolic activity against the compound. Examples of secondary reactions which provide no apparent benefit and are only encountered under laboratory conditions, such as the dechlorination of chloropurines by adenosine deaminase (), have been reported, and without further evidence it is more likely that this is the case for Pseudomonas sp. strain AAC. However, the discovery of three genes encoding proteins with this activity within the same organism suggests that the compound has a broader metabolic context, or that the genes coevolved from a single parent gene ().

INTRODUCTION

As stated above, among Pseudomonas sp. strain AAC proteins, three transaminases were identified with 12-aminododecanoic acid activity: , , and . The most promising candidate biocatalyst in terms of ease of protein production, solubility, and activity was , and here we report the subsequent characterization of that protein.