Crystal Structure of the E1 Component of the Escherichia coli 2-Oxoglutarate Dehydrogenase Multienzyme Complex

Abstract

The thiamine-dependent E1o component (EC 1.2.4.2) of the 2-oxoglutarate dehydrogenase complex catalyses a rate-limiting step of the tricarboxylic acid cycle (TCA) of aerobically respiring organisms. We describe the crystal structure of Escherichia coli E1o in its apo and holo forms at 2.6 Å and 3.5 Å resolution, respectively. The structures reveal the characteristic fold that binds thiamine diphosphate and resemble closely the α₂β₂ hetero-tetrameric E1 components of other 2-oxo acid dehydrogenase complexes, except that in E1o, the α and β subunits are fused as a single polypeptide. The extended segment that links the α-like and β-like domains forms a pocket occupied by AMP, which is recognised specifically. Also distinctive to E1o are N-terminal extensions to the core fold, and which may mediate interactions with other components of the 2-oxoglutarate dehydrogenase multienzyme complex. The active site pocket contains a group of three histidine residues and one serine that appear to confer substrate specificity and the capacity to accommodate the TCA metabolite oxaloacetate. Oxaloacetate inhibits E1o activity at physiological concentrations, and we suggest that the inhibition may allow coordinated activity within the TCA cycle. We discuss the implications for metabolic control in facultative anaerobes, and for energy homeostasis of the mammalian brain.

Introduction

For all eukaryotes, archaea, and most eubacteria, the metabolic “hub” for both catabolic and anabolic processes that provides key metabolic intermediates, and a vital resource of energy, is the citric acid/tricarboxylic acid (TCA) cycle, also known as the Krebs cycle. The route of metabolites through the cycle is affected by factors such as available carbon source and oxidative state (e.g. aerobic, anaerobic and oxidative stress).¹ Control within the cycle is exerted at several points, especially at the reactions that are far from equilibrium, such as those catalysed by isocitrate dehydrogenase² and the 2-oxoglutarate dehydrogenase (OGDH) multienzyme complex.³ The OGDH complex is largely rate-limiting in the TCA cycle in many species, and is a crucial nexus in the utilization of sugar and amino acids, which are “funnelled” via 2-oxoglutarate and oxidized to succinyl-CoA, releasing CO₂ and reducing NAD⁺ to NADH. The importance of the OGDH complex in highly active tissues is highlighted by its selective inactivation in the pathologies of several neurodegenerative disorders, particularly those associated with oxidative stress of mitochondria, such as Alzheimer's and Parkinson's diseases.4., 5., 6.

The OGDH complex is comprised of multiple copies of three types of component enzymes: a 2-oxoglutarate decarboxylase dependent on thiamine diphosphate (E1o, EC 1.2.4.2), a lipoylated succinyl transferase (E2o, EC 2.3.1.6), and a dihydrolipoyl dehydrogenase (E3, EC 1.8.1.4). The quaternary organisation of the OGDH complex ensures that the products of one reaction are efficiently shuttled to the next active site for the subsequent reaction.⁷ The three-stage catalytic mechanism of the OGDH multienzyme complex (as depicted in Figure 1(a)) is related to that of other 2-oxo acid dehydrogenase complexes (such as pyruvate dehydrogenase, PDH; and branched-chain 2-oxo acid dehydrogenases, BCDH) and extensive investigations into these complexes have also revealed corresponding similarities in their quaternary organisation.8., 9., 10.

In almost all instances, E1 and E3 are recruited into their multi-enzyme complexes through direct interaction with E2, which is an oligomeric enzyme with structurally distinct domains that are linked by flexible regions of polypeptide chain of 28–50 residues that are rich in Ala and Pro. In eukaryotes, however, an additional E2-like protein (referred variously as E3BP or protein X) is required to specifically bind E3. Although the organisation of the three component enzymes is crucial for the activity of the complex, various architectures have evolved for the dehydrogenases, though it is not clear if these differences affect function.⁷ Thus, the E2 assembles into either a 24-mer with octahedral symmetry or a 60-mer with icosahedral symmetry, depending on the organism. All BCDH, OGDH, and most PDH E2 from Gram-negative bacteria associate to form 24-mers,¹¹ whereas in eukaryotes and Gram-positive bacteria, the PDH E2 are icosahedral 60-mers.¹² The multiplicity of E2 subunits dictates the upper limit for the number of E1 and E3 that may join the complex, giving rise to ordered clusters of 5×10⁶ to 11 × 10⁶ Da.¹³ Puzzlingly, fewer E1o bind per E2o in the OGDH complex compared with the other 2-oxo acid dehydrogenase multienzyme complexes¹⁴ (Supplementary Data).

Structures determined by crystallography are available for each globular component of the OGDH multienzyme complex, with the single exception of E1o.15., 16., 17., 18. Moreover, at least one crystal structure is available for all other enzymes that constitute the TCA cycle. We present here the crystal structure and functional analyses of the Escherichia coli 2-oxoglutarate decarboxylase, revealing the molecular details of this last structurally characterised component of the TCA cycle. The structure reveals a homo-dimer in which the protomers have the recurring fold characteristic of thiamine-dependent enzymes; however, the E1o differs by the inclusion of specialized ligand-binding pockets, whose function we explore.