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Title:	Engineering of escherichia coli 2-oxoglutarate dehydrogenase complex with mechanistic and synthetic goals
Author:	Chakraborty, Joydeep
View Online:	njit-etd2019-041 (xv, 149 pages ~ 8.0 MB pdf)
Department:	Department of Chemistry and Environmental Science
Degree:	Doctor of Philosophy
Program:	Environmental Science
Document Type:	Dissertation
Advisory Committee:	Farinas, Edgardo Tabion (Committee co-chair) Jordan, Frank (Committee co-chair) Mitra, S. (Committee member) Kim, Yong Ick (Committee member) Li, Mengyan (Committee member)
Date:	2019-08
Keywords:	2-oxo acid dehydrogenase complexes Dihydrolipoamide acyltransferase E2 component Directed evolution Green chemistry Protein engineering Saturation mutagenesis
Availability:	Unrestricted
Abstract:	The Escherichia coli 2-oxoglutarate dehydrogenase complex (OGDHc) compromises multiple copies of three enzymes - 2-oxoglutarate dehydrogenase (E1o), dihydrolipoyl succinyltransferase (E2o), and dihydrolipoyl dehydrogenase (E3). OGDHc is found in the Krebs cycle and catalyzes the formation of the all-important succinyl-Coenzyme A (succinyl-CoA). OGDHc was engineered to understand the catalytic mechanism and optimized for chemical synthetic goals. Succinyl-CoA formation takes place within the catalytic domain of E2o via a transesterification reaction. The succinyl group from the thiol ester of S8-succinyldihydrolipoyl-E2o is transferred to the thiol group of CoA. Mechanistic studies were designed to investigate enzymatic transthioesterification. His375 and Asp374 was shown to be important in E2o. The magnitude of the rate acceleration provided by these residues suggests a role in stabilization of the symmetrical tetrahedral oxyanionic intermediate by formation of two hydrogen bonds, rather than in acid–base catalysis. Further evidence ruling out a role in acid–base catalysis is provided by saturation mutagenesis studies at His375 and substitutions to other potential hydrogen bond participants at Asp374. The rate constant for reductive succinylation of the E2o lipoyl domain (LDo) by E1o and 2-oxoglutarate (99 s-1) was approximately twofold larger than the rate constant for kcat (48 s-1) for the overall reaction (NADH production). It could be concluded that succinyl transfer to CoA and release of succinyl-CoA is the rate-limiting step. The results suggest a revised mechanism of catalysis for acyl transfer in the superfamily of 2-oxo acid dehydrogenase complexes, thus provide fundamental information regarding acyl-CoA formation, so important for several biological processes including post-translational succinylation of protein lysines. OGDHc was converted from a 2-oxoglutarate dehydrogenase to a 2-oxo aliphatic dehydrogenase complex by engineering consecutive components. 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 and several variants were identified for 2-OV activity. Variants also displayed activity for larger aliphatic substrates, which demonstrates the potential green synthetic utility.