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

Reactions

Yujeong Shim¹  Edgardo T. Farinas¹  Frank Jordan²  Xu Zhang²  Elena L. Guevara²  Hetal Patel²  Natalia Nemeria²  Joydeep Chakraborty³ 

[1]Department of Chemistry and Environmental Science, New Jersey Institute of Technology, Newark, NJ 07102, USA

[2]Department of Chemistry, Rutgers University, Newark, NJ 07103, USA

[3]Texas A&M Agrilife Research, Texas A&M University, College Station, TX 77843, USA

关键词: multienzyme complex;    site saturation mutagenesis;    catalysis;    thiamin diphosphate;    chemoenzymatic synthesis;    carboligation;   

DOI  :  10.3390/reactions3010011

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown