Discovery of a Two-Step Enzyme Cascade Converting Aspartate to Aminomalonate in Peptide Natural Product Biosynthesis

Aminomalonic acid (Ama) is found in various natural products and protein hydrolysates of multiple organisms, but the understanding of its biosynthetic origin remains largely limited. By exploiting a biosynthetic gene cluster for ribosomally synthesized and post-translationally modified peptides (RiPPs), which are rich sources of new enzyme chemistry, we identified a novel two-enzyme pathway for Ama biosynthesis. This biosynthetic pathway, mediated by an Fe(II)/2-oxoglutarate-dependent oxygenase (Fe(II)/2OG), SmaO, and an atypical Fe(II)-dependent histidine-aspartate (HD) domain enzyme, SmaX, converts aspartate (Asp) to β-hydroxyaspartic acid (Hya) intermediate and ultimately to Ama. These tandem enzymatic reactions─hydroxylation of the carbon next to an acid functional group and subsequent four-electron oxidative bond cleavage in α-hydroxy acid─are similar to those associated with other known HD domain oxygenases, PhnZ and TmpB. However, SmaX also exhibits unique features, such as C-C bond cleavage in α-hydroxycarboxylate using a single Fe cofactor, in contrast to the C-P bond cleavage using a mixed-valent diiron cofactor in PhnZ and TmpB. Bioinformatic analysis reveals that this two-enzyme cascade may be present in various biosynthetic pathways for peptide natural products, including RiPPs and nonribosomal peptides (NRPs). Collectively, our study demonstrates the presence of a novel Ama biosynthetic pathway and suggests its widespread distribution in peptide natural product biosynthesis.