Engineering 2-oxoglutarate dehydrogenase to a 2-oxo aliphatic dehydrogenase complex by optimizing consecutive components
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abstract
AbstractMultienzyme complexes have the potential for green catalysis of sequential reactions. The Escherichia coli 2oxoglutarate dehydrogenase complex (OGDHc) was converted from a 2oxoglutarate dehydrogenase to a 2oxo aliphatic dehydrogenase complex by engineering consecutive components. OGDHc catalyzes succinylCoA synthesis in the Krebs cycle. OGDHc is composed of three components: E1o, 2oxoglutarate 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. WtODGHc does not accept aliphatic substrates. E1o was previously engineered to accept a nonnatural aliphatic substrate, 2oxovalerate (2OV). E2o also required engineering to accept 2OV in the overall reaction. Hence, saturation mutagenesis libraries of E2o were screened for 2OV activity. E2oS333M, E2oH348F, E2oH348Q, and E2oH348Y were identified to show activity for 2OV in the reconstituted complex. Variants also displayed activity for larger aliphatic substrates.
