Zymophore identification enables the discovery of novel phenylalanine ammonia lyase enzymes

Abstract

The suite of biological catalysts found in Nature has the potential to contribute immensely to scientific advancements, ranging from industrial biotechnology to innovations in bioenergy and medical intervention. The endeavour to obtain a catalyst of choice is, however, wrought with challenges. Herein we report the design of a structure-based annotation system for the identification of functionally similar enzymes from diverse sequence backgrounds. Focusing on an enzymatic activity with demonstrated synthetic and therapeutic relevance, five new phenylalanine ammonia lyase (PAL) enzymes were discovered and characterised with respect to their potential applications. The variation and novelty of various desirable traits seen in these previously uncharacterised enzymes demonstrates the importance of effective sequence annotation in unlocking the potential diversity that Nature provides in the search for tailored biological tools. This new method has commercial relevance as a strategy for assaying the 'evolvability' of certain enzyme features, thus streamlining and informing protein engineering efforts.

Figure 1

The catalytic activity of phenylalanine ammonia lyase (PAL) enzymes as implicated in biosynthesis/biotherapeutics (a) and biocatalysis (b).

Figure 2

Flow diagram of the strategy employed to find new PAL enzymes for synthetic and medical applications.

Figure 3

The zymophore conferring PAL-specific reactivity and selectivity as identified from the co-crystal structure of the ammonia lyase from Anabaena variabilis (AvPAL) and its deamination product trans-cinnamate (PDB ID: 5LTM). The motif is shown on both structural overlay of the empty and occupied enzyme active sites and the sequence with each of the 19 amino acids given a number to mark its position.

Figure 4

Variation of specific activity (a), pH tolerance (b) and stability at 37 °C (c) among four new ammonia lyases from Streptomyces rimosus, Brevibacillus laterosporus, Planctomyces brasiliensis and Dictyostelium discoideum which could be isolated for investigation.

Figure 5

Preparative scale synthesis of L-m-methoxyphenylalanine (S)-2l by the newly discovered ammonia lyase from Planctomyces brasiliensis (PbPAL) and examples of antiviral and antidiabetic compounds 5 and 6 which can be synthesised from this building block.