Revealing the Structural Basis of Promiscuous Amine Transaminase Activity

By Fabian Steffen‐Munsberg, Clare Vickers, A. Thontowi, S. Schätzle, Tina Meinhardt, Maria Svedendahl Humble, Henrik Land, Per Berglund, U. Bornscheuer, M. Höhne. 2013

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Enzymes are nature’s tool to speed up biochemical reactions that would otherwise hardly proceed. As a result of the complexity of the metabolic network of living cells, enzymes have to fulfill partially competing demands. On the one hand, a high precision in terms of reaction and substrate specificity is often required. This is achieved, for example, by a fine-tuned interaction between the active site structure and the substrate, which forces a defined positioning of the substrates’ reactive groups relative to the catalytic residues. On the other hand, some enzymes have evolved with a relaxed substrate specificity, which is useful and economic in several metabolic tasks: For example, a single enzyme can thus break down various chemically similar nutrients or transform different poisonous compounds for their detoxification. The ability to catalyze more than one chemical transformation within a single active site, called promiscuity, is not only an important property of some enzymes, but it is also recognized as a key mechanism of evolution and thus facilitates adaptation of enzymes to new metabolic needs. Furthermore, the enzymes’ ability to take over more than one physiological function renders metabolism more plastic, even if the promiscuous “underground activities” are very weak. Substrate d] and reaction promiscuity are also often the reasons why enzymes can be applied in biocatalysis. Many fine chemicals that are synthesized by biocatalytic transformations are not produced in nature, but nevertheless, enzymes that can be applied in their syntheses have been identified. For example, pyruvate decarboxylase can be used for the production of (R)-phenylacetylcarbinol. Esterases and lipases are successfully used in organic synthesis for the preparation of various optically active alcohols, acids, and amines. Their natural function, however, might be totally different, as shown for pig liver esterase, which plays a role in signal transduction by ester cleavage of methylated and prenylated proteins. Still, for most enzymes their promiscuous potential has not yet been revealed. One reason is that, after the main activity and possible function has been identified, further in-depth characterization is often not performed. Furthermore, if the promiscuous activity is unrelated to the natural one, a general activity screen is very laborious. Therefore, the understanding of the molecular principles of promiscuity is a challenging frontier in biocatalysis, as the knowledge obtained can be used as a starting point for protein engineering or for the discovery of other promiscuous enzymes. In a recent study, we identified four crystal structures [protein database (PDB) codes: 3HMU, 3I5T, 3FCR, and 3GJU] of transaminases with unknown function, and we could show that these enzymes interconvert small amino acids, for example, alanine (1) and succinic semialdehyde (2, Scheme 1). Additionally, chiral amines can be transaminated, but with very different efficiencies that range from 0.4 to 80 % relative activity, compared with the transamination of 1 + 2 (see values given in Scheme 1). From these observations, we concluded that the conversion of amines is a type of substrate promiscuity, which is pronounced to varying degrees in these four enzymes. Transaminases showing this type of catalytic activity are designated amine transaminases (ATAs): In contrast to the ubiquitous a-amino acid transaminases, they accept substrates lack-

Subfields: Biochemistry, Materials Chemistry, Molecular Biology

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