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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 A and 3.5 A resolution, respectively. The structures reveal the characteristic fold that binds thiamine diphosphate and resemble closely the alpha(2)beta(2) hetero-tetrameric E1 components of other 2-oxo acid dehydrogenase complexes, except that in E1o, the alpha and beta subunits are fused as a single polypeptide. The extended segment that links the alpha-like and beta-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.

SUBMITTER: Frank RA

PROVIDER: S-EPMC7611002 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The region encompassing residues 401-413 on the E1 component of the pyruvate dehydrogenase multienzyme complex from Escherichia coli comprises a loop (the inner loop) which was not seen in the X-ray structure in the presence of thiamin diphosphate, the required cofactor for the enzyme. This loop is seen in the presence of a stable analogue of the pre-decarboxylation intermediate, the covalent adduct between the substrate analogue methyl acetylphosphonate and thiamin diphosphate, C2α-phosphonolactylthiamin diphosphate. It has been shown that the residue H407 and several other residues on this loop are required to reduce the mobility of the loop so electron density corresponding to it can be seen once the pre-decarboxylation intermediate is formed. Concomitantly, the loop encompassing residues 541-557 (the outer loop) appears to work in tandem with the inner loop and there is a hydrogen bond between the two loops ensuring their correlated motion. The inner loop was shown to: a) sequester the active center from carboligase side reactions; b) assist the interaction between the E1 and the E2 components, thereby affecting the overall reaction rate of the entire multienzyme complex; c) control substrate access to the active center. Using viscosity effects on kinetics it was shown that formation of the pre-decarboxylation intermediate is specifically affected by loop movement. A cysteine-less variant was created for the E1 component, onto which cysteines were substituted at selected loop positions. Introducing an electron spin resonance spin label and an (19)F NMR label onto these engineered cysteines, the loop mobility was examined: a) both methods suggested that in the absence of ligand, the loop exists in two conformations; b) line-shape analysis of the NMR signal at different temperatures, enabled estimation of the rate constant for loop movement, and this rate constant was found to be of the same order of magnitude as the turnover number for the enzyme under the same conditions. Furthermore, this analysis gave important insights into rate-limiting thermal loop dynamics. Overall, the results suggest that the dynamic properties correlate with catalytic events on the E1 component of the pyruvate dehydrogenase complex.

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Crystal structure of the E1 component of the Escherichia coli 2-oxoglutarate dehydrogenase multienzyme complex.

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