Investigation of Substrate Specificity in 5‐Keto‐4‐Deoxy‐Glucarate Dehydratase and 2‐Keto‐3‐Deoxy‐L‐Arabinonate Dehydratase

5-Keto-4-deoxy glucarate dehydratase and 2-keto-3-deoxy-L-arabinonate dehydratase are members of the dihydrodipicolinate synthase superfamily of aldolase class I enzymes. The enzymes are part of the oxidative nonphosphorylated metabolic pathways for the production of 2-ketoglutarate from glucuronic acid and arabinose, respectively. They carry out a C4 dehydration on their substrates, 5-keto-4-deoxy glucarate and 2-keto-3-deoxy-L-arabinonate, and produce the same product, ketoglutarate semialdehyde. While the enzymes are similar in structure and reaction mechanism, they exhibit a high substrate specificity and have only been reported to be active on their native substrate. In order to investigate the high substrate specificity, we aimed to generate promiscuous variants of both enzymes that can accept multiple substrates and investigate the evolutionary development of the substrate specificity in the superfamily. Ancestral sequence reconstruction and GFN2-xTB calculations were used to develop candidates for further analysis of substrate scope. Both approaches yielded multiple promiscuous dehydratases with activity on both substrates.