Arrangement and symmetry of the fungal E3BP-containing core of the pyruvate dehydrogenase complex

Abstract

The pyruvate dehydrogenase complex (PDC) is a multienzyme complex central to aerobic respiration, connecting glycolysis to mitochondrial oxidation of pyruvate. Similar to the E3-binding protein (E3BP) of mammalian PDC, PX selectively recruits E3 to the fungal PDC, but its divergent sequence suggests a distinct structural mechanism. Here, we report reconstructions of PDC from the filamentous fungus Neurospora crassa by cryo-electron microscopy, where we find protein X (PX) interior to the PDC core as opposed to substituting E2 core subunits as in mammals. Steric occlusion limits PX binding, resulting in predominantly tetrahedral symmetry, explaining previous observations in Saccharomyces cerevisiae. The PX-binding site is conserved in (and specific to) fungi, and complements possible C-terminal binding motifs in PX that are absent in mammalian E3BP. Consideration of multiple symmetries thus reveals a differential structural basis for E3BP-like function in fungal PDC.

Structure of the native pyruvate dehydrogenase complex reveals the mechanism of substrate insertion

“…However, we were not able to unambiguously assign the individual E1p and/or E3 components into a complete model due to the conformational flexibility and compositional heterogeneity of the outer shell (Supplementary Fig. 4a ), which is not geometrically precise and does not exactly follow the symmetry of the E2p core 6 , 8 , 9 , 28 , 29 , 40 .…”

“…It is structurally and functionally related to the other oxoacid dehydrogenase complexes: α-ketoglutarate dehydrogenase complex (E1o, E2o, and E3) and branched-chain α-ketoacid dehydrogenase complex (E1b, E2b, and E3), which all have individual E1 and E2 subunits but share a common E3 component 6 . Mitochondrial PDHc contains an E3-binding protein (protein X), and its activity is additionally regulated by E1 kinases and phosphatases 7 – 9 . In the first reaction step catalyzed by E1p, pyruvate is decarboxylated using thiamine pyrophosphate (TPP, also often referred to as TDP or ThDP for thiamine/vitamin B1 diphosphate) as a cofactor and the resulting hydroxyethyl-TPP then reacts with lipoamide (oxidized lipoic acid attached to a lysine residue of E2p 10 ) to produce S-acetyldihydrolipoamide and recover the TPP.…”

“…The size, composition, and symmetry of the oxoacid dehydrogenase complexes vary in different organisms, but the structural and functional core is always a dihydrolipoyl acyltransferase (E2). It provides binding sites for the other enzymes (together with the E3-binding proteins in mitochondria 9 , 11 ) and conveys reaction intermediates between spatially distant active sites in the complex. The covalent substrate channeling conducted by E2 (and E3-binding protein in eukaryotes) tightly couples the individual reactions and increases the overall rate of the whole complex 1 , 3 , 6 , 7 (Fig.…”

Singapore Grouper Iridovirus Induces Glucose Metabolism in Infected Cells by Activation of Mammalian Target of Rapamycin Signaling

“…It has been reported that exogenous glucose uptake into cells is mediated by glucose transporters (GLUTs), and glucose metabolism is catalyzed by several key enzymes, including HKs and PDHX ( Gershon et al, 2013 ; Adeva-Andany et al, 2016 ; Forsberg et al, 2020 ). To determine whether SGIV infection activated glycolysis, the transcription levels of HK2, PDHX, GLUT1, and GLUT2 were firstly examined in vivo by qPCR during SGIV infection.…”

“…For example, hexokinases (HKs), which convert glucose to glucose 6-phosphate, is the first and rate-limiting enzyme in glycolytic pathway and the HK2 isoform is a key mediator of aerobic glycolysis ( Wolf et al, 2011 ; Gershon et al, 2013 ). Pyruvate dehydrogenase complex (PDHX), which provide acetyl-CoA for the TCA cycle, is a multienzyme complex central to aerobic respiration, connecting glycolysis to mitochondrial oxidation of pyruvate ( Forsberg et al, 2020 ).…”