Engineering the 2-Oxoglutarate Dehydrogenase Complex to Understand Catalysis and Alter Substrate Recognition
Document Type
Article
Publication Date
3-1-2022
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.
Identifier
85168251406 (Scopus)
Publication Title
Reactions
External Full Text Location
https://doi.org/10.3390/reactions3010011
e-ISSN
2624781X
First Page
139
Last Page
159
Issue
1
Volume
3
Grant
1402675
Fund Ref
National Science Foundation
Recommended Citation
Chakraborty, Joydeep; Nemeria, Natalia; Shim, Yujeong; Zhang, Xu; Guevara, Elena L.; Patel, Hetal; Farinas, Edgardo T.; and Jordan, Frank, "Engineering the 2-Oxoglutarate Dehydrogenase Complex to Understand Catalysis and Alter Substrate Recognition" (2022). Faculty Publications. 3088.
https://digitalcommons.njit.edu/fac_pubs/3088
