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Title:	Escherichia coli 2-oxoglutarate dehydrogenase multienzyme complex: e1 and e2 substrate specificty, e1 carboligase activity, and e2 interchain succinyl transfer
Author:	Shim, Da Jeong
View Online:	njit-etd2013-090 (xix, 105 pages ~ 4.2 MB pdf)
Department:	Department of Chemistry and Environmental Science
Degree:	Doctor of Philosophy
Program:	Chemistry
Document Type:	Dissertation
Advisory Committee:	Farinas, Edgardo Tabion (Committee co-chair) Jordan, Frank (Committee co-chair) Mitra, S. (Committee member) Wei, Liping (Committee member) Huang, Haidong (Committee member)
Date:	2013-05
Keywords:	Saturation mutagenesis 2-oxoglutarate dehydrogenase Screening Enzyme kinetic Carboligase reaction Green chemistry
Availability:	Unrestricted
Abstract:	Escherichia coli (E. coli) 2-oxoglutarate dehydrogenase multienzyme complex (OGDHc) contains three components: a thiamin di phosphate (ThD P) dependent 2-oxogl utarate dehydrogenase (E1 o), a di hydrol i poylsucci nyl transferase (E2o), and a di hydrol i poyl dehydrogenase (E3). The first two components carry out the principal reactions for succinyl CoA formation while the third one reoxidizes dihydrolipoamide to lipoamide. This mechanism is similar to other 2-oxoacid dehydrogenase complexes, including pyruvate dehydrogenases (PDHc) and branched-chain dehydrogenases. E1o of the E. coli OGDHc was engineered to accept unnatural substrates. The natural substrate for E1 o is 2-oxogl utarate (2-OG) and the enzyme was engineered to accept substrates lacking the 5-carboxylate group, 2-oxovalerate (2-OV). E1o was subjected to saturation mutagenesis at H is260 and H is298. The H is298Asp E1 o variant was identified in the screen to accept the unnatural substrate. In addition, it was discovered that His260 is required for substrate recognition, but His298 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, E1 p for pyruvate but E2o for 2-OG. Although His298Asp E1o accepted 2-OV, reconstitution of the variant with E2o and E3 did not generate NADH in the overall reaction using 2-OV. Hence, the reaction may be hindered in the E2o component. E2o consists of an amino-terminal lipoyl domain (E2oLD, 12 kDa), followed by a peripheral subunit binding domain (4 kDa) and a succinyltransferase domain (E2oCD, 28 kDa). There are two possibilities for the failure to form NADH. Reductive acylation is not occurring in the E2oLD or acyl transfer to CoA is not taking place in E2oCD. His298Asp E1o, E2oLD, and 2-OV form butyrylated E2oLD, which was shown by electrospray ionization mass spectrometry. Therefore, the E2oCD domain necessitates optimization to produce acyl-CoA derivatives. Succinyl transfer to the CD domain may occur through an intrachain or interchain pathway. The E2oLD and E2o with a lysine to alanine substitution at position 43 (E2oK43A) were created by site directed mutagenesis. It is clearly shown that E2oLD was capable of rescuing the crippled E2oK43A variant by measuring the NADH production in the overall reaction. Therefore, an interchain mechanism is likely between two different E2o subunits. ThDP-dependent enzymes have the potential to be used for chemical synthesis. These enzymes share a common feature in that they catalyze carboligase reactions. E1o catalyzed carboligation products with a variety of substrates and acceptors that vary in the size and functional groups. Structures of the products were confirmed with NMR. In addition, high enantiomeric excess (ee) values were found for the products as shown by chiral gas chromatography and CD spectroscopy. Finally, it was shown that E1o is capable of forming stable esters. This is important because when the carboligase reactions produce 0-ketoacids, these products are unstable and prone to decarboxylation.