The first component (E1o) of the Escherichia coli 2-oxoglutarate dehydrogenase complex (OGDHc) was engineered to accept substrates lacking the 5-carboxylate group by subjecting H260 and H298 to saturation mutagenesis. Apparently, H260 is required for substrate recognition, but H298 could be replaced by hydrophobic residues of similar molecular volume. To interrogate whether the second component would enable synthesis of acyl-coenzymeA derivatives, hybrid complexes consisting of recombinant components of OGDHc (o) and pyruvate dehydrogenase (p) enzymes were constructed, suggesting that a different component is the ‘gatekeeper’ for specificity for these two multienzyme complexes in bacteria, E1p for pyruvate, but E2o for 2-oxoglutarate.

The potential of thiamin diphosphate (ThDP)-dependent enzymes to catalyze C-C bond forming (carboligase) reactions with high enantiomeric excess has been recognized for many years. Here we report the application of the E1 component of the Escherichia coli 2-oxoglutarate dehydrogenase multienzyme complex in the synthesis of chiral compounds with multiple functional groups in good yield and high enantiomeric excess, by varying both the donor substrate (different 2-oxo acids) and the acceptor substrate (glyoxylate, ethyl glyoxylate and methyl glyoxal). Major findings include the demonstration that the enzyme can accept 2-oxovalerate and 2-oxoisovalerate in addition to its natural substrate 2-oxoglutarate, and that the tested acceptors are also acceptable in the carboligation reaction, thereby very much expanding the repertory of the enzyme in chiral synthesis.

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The substrate specificity of the 2‐oxoglutarate dehydrogenase multienzyme complex (OGDH) was engineered to accept unnatural substrates. OGDH catalyzes the rate limiting step in the citric acid cycle. It contains three components: a thiamine diphosphate dependent oxoglutarate decarboxylase (E1), a dihydrolipoylsuccinyl transferase (E2), and a dihydrolipoyl dehydrogenase (E3). The crystal structure, kinetic studies, and site directed mutagenesis analysis of the E1 component indicate that His260 and His298 are important for recognizing the distal carboxylate of 2‐oxoglutarate. Saturation mutagenesis libraries of His260, His298, and His260/His298 were constructed and screened for activity towards 2‐oxovalerate. Several E1 variants were isolated that are active towards 2‐oxovalerate, and the kinetic parameters were determined. In addition, circular dichroism investigations were performed that identified the formation of the pre‐decarboxylation thiamin‐bound intermediate via formation of the 1’, 4’‐imino tautomer of ThDP. The activity of the overall complex on reconstitution with the E2–E3 sub‐complex shows that substrate specificity is controlled at both the E1 and E2 levels.