Diverse functional evolution of serine decarboxylases: identification of two novel acetaldehyde synthases that uses hydrophobic amino acids as substrates

Abstract

Background: Type II pyridoxal 5'-phosphate decarboxylases are an important group of phylogenetically diverse enzymes involved in amino acid metabolism. Within plants, this group of enzymes is represented by aromatic amino acid decarboxylases, glutamate decarboxylases and serine decarboxylases. Additional evolutionary divergence of plant aromatic amino acid decarboxylases has resulted in further subcategories with distinct substrate specificities and enzymatic activities. Despite shared homology, no such evolutionary divergence has been characterized within glutamate decarboxylases or serine decarboxylases (SDC).

Results: Comparative analysis of two previously characterized serine decarboxylase-like (SDC-like) enzymes demonstrates distinct substrate specificities despite their highly conserved primary sequence. The alternate substrate preference of these homologous SDC-like proteins indicated that functional divergence might have occurred with in SDC-like proteins. In an effort to identify additional SDC-like functional divergence, two uncharacterized SDC-like enzymes were recombinantly expressed and characterized.

Conclusions: An extensive biochemical analysis of two serine decarboxylases-like recombinant proteins led to an interesting discovery; both proteins catalyze the formation of acetaldehyde derivatives from select hydrophobic amino acids substrates. Specifically, Medicago truncatula [GenBank: XP_003592128] and Cicer arietinum [GenBank: XP_004496485] catalyze the decarboxylation and oxidative deamination of phenylalanine, methionine, leucine and tryptophan to generate their corresponding acetaldehydes. The promiscuous aldehyde synthase activity of these proteins yields novel products of 4-(methylthio) butanal, 3-methylbutanal (isovaleraldehyde) and indole-3-acetaldehyde from methionine, leucine and tryptophan respectively. A comparative biochemical analysis of the Medicago truncatula and Cicer arietinum enzymes against two previously characterized SDC-like enzymes further emphasizes the unusual substrate specificity and activity of these novel aldehyde synthases. Due to the strong substrate preference towards phenylalanine, it is likely that both enzymes function as phenylacetaldehyde synthesis in vivo. However, due to their significant sequence divergence and unusual substrate promiscuity these enzymes are functionally and evolutionary divergent from canonical phenylacetaldehyde synthesis enzymes. This work further elaborates on the functional complexity of plant type II PLP decarboxylases and their roles in secondary metabolite biosynthesis.

Figure 1

HPLC-EC detection of indole-3-acetaldehyde generated in MtAAS and tryptophan reaction mixtures. (Chromatograms A-F) Y-axis represents the output in microamps and the x-axis represents retention time. Chromatograms (A-C) illustrate the indole-3-acetaldehyde (the major broad peak) formed in MtAAS and tryptophan reaction mixtures after 5 min, 20 min and 40 min incubation, respectively. Chromatograms (D-F) illustrate the indole-3-ethanol (tryptophol) formed in borohydride reduced MtAAS and tryptophan reaction mixtures after 5 min, 20 min and 40 min incubation, respectively. Chromatogram (G) shows the detection of authentic indole-3-ethanol standard.

Figure 2

GCMS analysis of authentic phenylacetaldehyde and MtAAS/CaAAS phenylalanine reaction products. (A) illustrates the elution and select ion monitoring of authentic phenylacetaldehyde. (B) illustrates the elution and select ion monitoring of the enzymatic product generated from MtAAS and phenylalanine. (C) illustrates the elution and select ion monitoring of the enzymatic product generated from MtAAS and phenylalanine.

Figure 3

Relative activities of aromatic amino acid decarboxylase (AAAD) and aromatic acetaldehyde synthases (AAS).

Figure 4

Intersection of the MtAAS and CaAAS enzymes and the proposed tryptophan dependent indole-3-pyruvic acid auxin biosynthetic pathway.