Engineering 2-oxoglutarate dehydrogenase to a 2-oxo aliphatic dehydrogenase complex by optimizing consecutive components

Multienzyme complexes have the potential for green catalysis of sequential reactions. The Escherichia coli 2-oxoglutarate dehydrogenase complex (OGDHc) was converted from a 2-oxoglutarate dehydrogenase to a 2-oxo aliphatic dehydrogenase complex by engineering consecutive components. OGDHc catalyzes succinyl-CoA synthesis in the Krebs cycle. OGDHc is composed of three components: E1o, 2-oxoglutarate dehydrogenase; E2o, dihydrolipoylsuccinyl transferase; E3, dihydrolipoyl dehydrogenase. There are three substrate checkpoints. One is in E1o and two in E2o. OGDHc was reprogrammed to accept alternative substrates by evolving the E1o and E2o components. Wt-ODGHc does not accept aliphatic substrates. E1o was previously engineered to accept a non-natural aliphatic substrate, 2-oxovalerate (2-OV). E2o also required engineering to accept 2-OV in the overall reaction. Hence, saturation mutagenesis libraries of E2o were screened for 2-OV activity. E2o-S333M, E2o-H348F, E2o-H348Q, and E2o-H348Y were identified to show activity for 2-OV in the reconstituted complex. Variants also displayed activity for larger aliphatic substrates.