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The Jordan group's interest center on: E. coli (ec) and human (h) pyruvate dehydrogenase (PDHc), 2-oxoglutarate dehydrogenase (OGDHc) and 2-oxoadipate dehydrogenase (OADHc) complexes; and on 1-deoxy-D-xylulose-5-phosphate synthase (DXPS). The most important findings were: (1) the first components of OGDHc and OADHc and ecDXPS aerobically generated the ThDP-enamine radical species in “off-pathway” mechanism. (2) The pre-steady-state rate constants for conversion of pyruvate to all intermediates were determined on the entire E. coli PDHc. (3) The hydrogen–deuterium exchange MS (HDX-MS) method provided a snapshot of (i) the conformational dynamics of the full-length E. coli DXPS; (ii) interaction loci of the ecE1p-ecE2p subcomplex; (iii) the unique inter-component interaction sites in the hE1o-hE2o subcomplex, also complemented by chemical cross-linking MS. (4) Protein engineering using site saturation mutagenesis was applied to the first and second components of the ecOGDHc and led to formation of acyl-Coenzyme A with a variety acyl groups.

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.

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 with hydrophobic residues of similar molecular volume. To interrogate whether the second component would allow synthesis of acyl-coenzyme A 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.