Expanding the substrate scope of C-N lyases by homologue discovery.

Abstract

The aspartase/fumarase superfamily is a group of homologous enzymes that promote the reversible elimination of functional groups from succinyl-containing compounds, typically yielding fumarate as the common product. Over the past 50 years, members of this superfamily have continuously demonstrated their power and significance as biocatalysts. This is exemplified by ethylenediamine-N,N-disuccinic acid (EDDS) lyase, which was shown to have an extraordinary amine scope, enabling the production of a wide variety of N-substituted aspartic acids. In this work, we used this enzyme as a starting point for a homology-based strategy to expand the biocatalytic toolbox of C-N bond-forming enzymes. We selected 13 enzymes for biochemical characterization, and identified several EDDS-lyase homologues that can accept L-amino acids as substrates in the hydroamination of fumarate to produce the corresponding aminopolycarboxylic acids. Lastly, we carried out a sequence similarity network analysis of the aspartase/fumarase superfamily, which suggests that EDDS lyase and its homologues may represent a distinct isofunctional subfamily, laying the foundations for future enzyme discovery and engineering campaigns.