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

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.

The importance of thiamine (vitamin B1) in humans

“…Literature data indicate that more than 20 different thiamine pyrophosphate-dependent enzymes are known, most of which are found in prokaryotes: transketolase [ 16 ], phosphonopyruvate decarboxylase [ 17 ], pyruvate dehydrogenase complex [ 18 ], branched-chain amino acid enzyme [ 19 ], phosphoketolase [ 20 ], benzoylformate decarboxylase [ 21 ], pyruvate decarboxylase [ 22 ], 2-oxoglutarate dehydrogenase complex [ 23 ], sulfopyruvate decarboxylase [ 24 ], pyruvate ferredoxinoxidoreductase [ 25 ], phenylpyruvate decarboxylase [ 26 ], 2-hydroxyphytanoyl-CoA lyase [ 27 ], acetohydroxyacid synthases [ 28 ], glyoxylate carboligase [ 29 ], and others.…”

Strategies on Designing Thiamine Analogues for Inhibition of Thiamine Diphosphate (ThDP)-dependent Enzymes: Systematic Investigation through Scaffold Switching and C2-Functionalisation

“…2,3 The remainder consists of free thiamine I, thiamine monophosphate III (ThMP), thiamine triphosphate IV (ThTP), adenosine thiamine diphosphate and adenosine thiamine triphosphate. ThDP (the bioactive form of I) is a coenzyme of ThDP-dependent enzymes, 4 an extensive family of enzymes that includes pyruvate dehydrogenase complex E1subunit 5,6 (PDH E1), pyruvate decarboxylase 6 (PDC), pyruvate oxidase 7 (PO), 1-deoxy-D-xylulose 5phosphate synthase [7][8][9][10][11][12][13] (DXPS) and oxoglutarate dehydrogenase complex E1-subunit 14,15 (OGDH E1) (Figure 1b). A general mechanism is outlined in Figure 1a in which a catalytically active ThDP-ylide V (formed by deprotonation at C2 of the thiazolium ring) chemically converts the substrate and regenerates itself upon product release in an iterative cycle.…”

Strategies on Designing Thiamine Analogues for Inhibition of Thiamine Diphosphate (ThDP)-dependent Enzymes: Systematic Investigation through Scaffold Switching and C2-Functionalisation

“…2,22 The remainder consists of free thiamine I, thiamine monophosphate III (ThMP), thiamine triphosphate IV (ThTP), adenosine thiamine diphosphate and adenosine thiamine triphosphate. ThDP (the bioactive form of I) is a coenzyme of ThDP-dependent enzymes, 3 an extensive family of enzymes that includes pyruvate dehydrogenase complex E1-subunit 4,5 (PDH E1), pyruvate decarboxylase 6 (PDC), pyruvate oxidase 7 (PO), 1-deoxy-Dxylulose 5-phosphate synthase [8][9][10][11][12] (DXPS) and oxoglutarate dehydrogenase complex E1-subunit 13,14 (OGDH E1) (Figure 1b). A general mechanism is outlined in Figure 1a in which a catalytically active ThDP-ylide V (formed by deprotonation at C2 of the thiazolium ring) chemically converts the substrate and regenerates itself upon product release in an iterative cycle.…”