Pyruvate dehydrogenase complex deficiency is linked to regulatory loop disorder in the αV138M variant of human pyruvate dehydrogenase

Matthew J Whitley,Palaniappa Arjunan,Natalia S Nemeria,Lioubov G Korotchkina,Yun-Hee Park,Mulchand S Patel,Frank Jordan,William Furey

doi:10.1074/jbc.ra118.003996

Abstract

The pyruvate dehydrogenase multienzyme complex (PDHc) connects glycolysis to the tricarboxylic acid cycle by producing acetyl-CoA via the decarboxylation of pyruvate. Because of its pivotal role in glucose metabolism, this complex is closely regulated in mammals by reversible phosphorylation, the modulation of which is of interest in treating cancer, diabetes, and obesity. Mutations such as that leading to the αV138M variant in pyruvate dehydrogenase, the pyruvate-decarboxylating PDHc E1 component, can result in PDHc deficiency, an inborn error of metabolism that results in an array of symptoms such as lactic acidosis, progressive cognitive and neuromuscular deficits, and even death in infancy or childhood. Here we present an analysis of two X-ray crystal structures at 2.7-Å resolution, the first of the disease-associated human αV138M E1 variant and the second of human wildtype (WT) E1 with a bound adduct of its coenzyme thiamin diphosphate and the substrate analogue acetylphosphinate. The structures provide support for the role of regulatory loop disorder in E1 inactivation, and the αV138M variant structure also reveals that altered coenzyme binding can result in such disorder even in the absence of phosphorylation. Specifically, both E1 phosphorylation at αSer-264 and the αV138M substitution result in disordered loops that are not optimally oriented or available to efficiently bind the lipoyl domain of PDHc E2. Combined with an analysis of αV138M activity, these results underscore the general connection between regulatory loop disorder and loss of E1 catalytic efficiency.

Highlights

The pyruvate dehydrogenase multienzyme complex (PDHc) connects glycolysis to the tricarboxylic acid cycle by producing acetyl-CoA via the decarboxylation of pyruvate
Mutations such as that leading to the ␣V138M variant in pyruvate dehydrogenase, the pyruvate-decarboxylating PDHc E1 component, can result in PDHc deficiency, an inborn error of metabolism that results in an array of symptoms such as lactic acidosis, progressive cognitive and neuromuscular deficits, and even death in infancy or childhood
Regarding the WT–AcPhi E1 structure, there have been no similar ligand complex structures reported for any other PDHc, regardless of species, apart from our own earlier report of the E. coli PDHc E1-phosphonolactylthiamin diphosphate complex [20]

Summary

Effects of a pathogenic mutation in pyruvate dehydrogenase

Pyruvate is the native decarboxylation substrate, whereas AcPhi is the inhibitory substrate analogue used in this study. B, representative omit mFo Ϫ DFc electron density contoured at 3␴ for the ThDP:AcPhi adduct in one binding site of WT human E1. Mutations resulting in PDHc deficiency can be found in the genes coding for the three main PDHc enzymatic components, but most are found in the pyruvate-decarboxylating E1 component. Human PDHc E1 is an ␣2␤2 heterotetramer that has a molecular mass of 150 kDa. Human PDHc E1 is an ␣2␤2 heterotetramer that has a molecular mass of 150 kDa Its function requires both the coenzyme ThDP and magnesium ions. In the present work we examine the structural basis for impaired function in the pathogenic ␣V138M variant of human PDHc E1 [5, 6]. The ␣V138M E1 structure, along with the structure of WT–AcPhi E1, underscores the connection between regulatory loop disorder and reduced function in this enzyme

Results

PDB code

Discussion

Expression and purification of human PDHc proteins

Enzyme activity measurements