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

Joydeep Chakraborty,Edgardo T. Farinas,Xu Zhang,Yujeong Shim,Elena L. Guevara,Natalia Nemeria,Frank Jordan,Hetal Patel

doi:10.3390/reactions3010011

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.

Highlights

According to site saturation mutagenesis (SSM) studies, the His375Trp E2o substitution led to an oxoglutarate dehydrogenase complex (OGDHc) activity of about 60% compared to wild-type E2o, arguing strongly against the acid–base role of His375 [106]. This conclusion is consistent with data reported earlier from McLeish’s group on the thiamin diphosphate (ThDP)-dependent enzyme benzoylformate decarboxylase (BFDC), where SSM studies on the active site His281 led to His281Phe and His281Trp variants with significant remaining activity compared to the wild-type enzyme, arguing against an acid–base role for His281 [18]
The major focus of the current review was to explore the power of protein engineering methods to investigate active site catalysis and further expansion of the substrate range of multienzyme complexes
A thorough understanding of both substrate binding and catalysis is essential in order to implement the complex for chemoenzymatic synthetic purposes

Summary

Introduction

We were interested in the engineering of the E. coli E1o active centers to accept substrates lacking the 5-carboxylate group, such as 2-oxovaleric acid (2-OV, Figure 2, top), by constructing saturation mutagenesis libraries at the E1o active site residues suggested to interact with the distal carboxylate of 2-OG [39,68]. The single-site substitution at the highly conserved His375, Asp374 and Thr323 from the E. coli E2o active center revealed that His375 and Asp374 but not Thr323 are catalytically the most important residues for succinyl transfer in both the physiological and reverse directions (see Figure 5B for steady-state kinetic data) [106].

Results

Conclusion