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10.3390/reactions3010011
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Review

Engineering the 2-Oxoglutarate Dehydrogenase Complex to Understand Catalysis and Alter Substrate Recognition

by
Joydeep Chakraborty
1,2,*,
Natalia Nemeria
3,
Yujeong Shim
2,
Xu Zhang
3,
Elena L. Guevara
3,
Hetal Patel
3,
Edgardo T. Farinas
2,* and
Frank Jordan
3,*
1
Texas A&M Agrilife Research, Texas A&M University, College Station, TX 77843, USA
2
Department of Chemistry and Environmental Science, New Jersey Institute of Technology, Newark, NJ 07102, USA
3
Department of Chemistry, Rutgers University, Newark, NJ 07103, USA
*
Authors to whom correspondence should be addressed.
Reactions 2022, 3(1), 139-159; https://doi.org/10.3390/reactions3010011
Submission received: 11 October 2021 / Revised: 21 January 2022 / Accepted: 28 January 2022 / Published: 1 February 2022
(This article belongs to the Special Issue Feature Papers in Reactions in 2021)
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Abstract

The E. coli 2-oxoglutarate dehydrogenase complex (OGDHc) is a multienzyme complex in the tricarboxylic acid cycle, consisting of multiple copies of three components, 2-oxoglutarate dehydrogenase (E1o), dihydrolipoamide succinyltransferase (E2o) and dihydrolipoamide dehydrogenase (E3), which catalyze the formation of succinyl-CoA and NADH (+H+) from 2-oxoglutarate. This review summarizes applications of the site saturation mutagenesis (SSM) to engineer E. coli OGDHc with mechanistic and chemoenzymatic synthetic goals. First, E1o was engineered by creating SSM libraries at positions His260 and His298.Variants were identified that: (a) lead to acceptance of substrate analogues lacking the 5-carboxyl group and (b) performed carboligation reactions producing acetoin-like compounds with good enantioselectivity. Engineering the E2o catalytic (core) domain enabled (a) assignment of roles for pivotal residues involved in catalysis, (b) re-construction of the substrate-binding pocket to accept substrates other than succinyllysyldihydrolipoamide and (c) elucidation of the mechanism of trans-thioesterification to involve stabilization of a tetrahedral oxyanionic intermediate with hydrogen bonds by His375 and Asp374, rather than general acid–base catalysis which has been misunderstood for decades. The E. coli OGDHc is the first example of a 2-oxo acid dehydrogenase complex which was evolved to a 2-oxo aliphatic acid dehydrogenase complex by engineering two consecutive E1o and E2o components.
Keywords: multienzyme complex; site saturation mutagenesis; catalysis; thiamin diphosphate; chemoenzymatic synthesis; carboligation multienzyme complex; site saturation mutagenesis; catalysis; thiamin diphosphate; chemoenzymatic synthesis; carboligation

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MDPI and ACS Style

Chakraborty, J.; Nemeria, N.; Shim, Y.; Zhang, X.; Guevara, E.L.; Patel, H.; Farinas, E.T.; Jordan, F. Engineering the 2-Oxoglutarate Dehydrogenase Complex to Understand Catalysis and Alter Substrate Recognition. Reactions 2022, 3, 139-159. https://doi.org/10.3390/reactions3010011

AMA Style

Chakraborty J, Nemeria N, Shim Y, Zhang X, Guevara EL, Patel H, Farinas ET, Jordan F. Engineering the 2-Oxoglutarate Dehydrogenase Complex to Understand Catalysis and Alter Substrate Recognition. Reactions. 2022; 3(1):139-159. https://doi.org/10.3390/reactions3010011

Chicago/Turabian Style

Chakraborty, Joydeep, Natalia Nemeria, Yujeong Shim, Xu Zhang, Elena L. Guevara, Hetal Patel, Edgardo T. Farinas, and Frank Jordan. 2022. "Engineering the 2-Oxoglutarate Dehydrogenase Complex to Understand Catalysis and Alter Substrate Recognition" Reactions 3, no. 1: 139-159. https://doi.org/10.3390/reactions3010011

APA Style

Chakraborty, J., Nemeria, N., Shim, Y., Zhang, X., Guevara, E. L., Patel, H., Farinas, E. T., & Jordan, F. (2022). Engineering the 2-Oxoglutarate Dehydrogenase Complex to Understand Catalysis and Alter Substrate Recognition. Reactions, 3(1), 139-159. https://doi.org/10.3390/reactions3010011

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