CryoEM analysis of the essential native UDP-glucose pyrophosphorylase from Aspergillus nidulans reveals key conformations for activity regulation and function

Abstract

Invasive aspergillosis is one of the most serious clinical invasive fungal infections, resulting in a high case fatality rate among immunocompromised patients. The disease is caused by saprophytic molds in the genus Aspergillus , including Aspergillus fumigatus , the most significant pathogenic species. The fungal cell wall, an essential structure mainly composed of glucan, chitin, galactomannan, and galactosaminogalactan, represents an important target for the development of antifungal drugs. UDP (uridine diphosphate)-glucose pyrophosphorylase (UGP) is a central enzyme in the metabolism of carbohydrates that catalyzes the biosynthesis of UDP-glucose, a key precursor of fungal cell wall polysaccharides. Here, we demonstrate that the function of UGP is vital for Aspergillus nidulans ( An UGP). To understand the molecular basis of An UGP function, we describe a cryoEM structure (global resolution of 3.5 Å for the locally refined subunit and 4 Å for the octameric complex) of a native An UGP. The structure reveals an octameric architecture with each subunit comprising an N-terminal α-helical domain, a central catalytic glycosyltransferase A-like (GT-A-like) domain, and a C-terminal (CT) left-handed β-helix oligomerization domain. An UGP displays unprecedented conformational variability between the CT oligomerization domain and the central GT-A-like catalytic domain. In combination with activity measurements and bioinformatics analysis, we unveil the molecular mechanism of substrate recognition and specificity for An UGP. Altogether, our study not only contributes to understanding the molecular mechanism of catalysis/regulation of an important class of enzymes but also provides the genetic, biochemical, and structural groundwork for the future exploitation of UGP as a potential antifungal target. IMPORTANCE Fungi cause diverse diseases in humans, ranging from allergic syndromes to life-threatening invasive diseases, together affecting more than a billion people worldwide. Increasing drug resistance in Aspergillus species represents an emerging global health threat, making the design of antifungals with novel mechanisms of action a worldwide priority. The cryoEM structure of UDP (uridine diphosphate)-glucose pyrophosphorylase (UGP) from the filamentous fungus Aspergillus nidulans reveals an octameric architecture displaying unprecedented conformational variability between the C-terminal oligomerization domain and the central glycosyltransferase A-like catalytic domain in the individual protomers. While the active site and oligomerization interfaces are more highly conserved, these dynamic interfaces include motifs restricted to specific clades of filamentous fungi. Functional study of these motifs could lead to the definition of new targets for antifungals inhibiting UGP activity and, thus, the architecture of the cell wall of filamentous fungal pathogens.

Architecture, Function, Regulation, and Evolution of α-Glucans Metabolic Enzymes in Prokaryotes

“…396 (ii) base-to-base dimerization allowing tetramerization as observed in the AGPase from E. coli, 188 (iii) side-by-side parallel trimerization as in the case of GlmU from E. coli, 298 and (iv) top-to-top dimerization allowing octamerization as in eukaryotic UGPases. 397,398 5.1.1.1. ADP-Glucose Pyrophosphorylase.…”

“…413,414 UGPases are present in all kingdoms of life. 389 UGPase protomers comprise a canonical NSPase domain, with or without a C-terminal LβH domain, showing diverse forms of oligomerization, from monomers 415,416 to octamers 398,417,418 (Figures 10 and 11).…”

“…UDP-glucose was the first nucleotide sugar discovered and arguably the most extended glucose donor in all metabolic pathways. , UGPases are present in all kingdoms of life . UGPase protomers comprise a canonical NSPase domain, with or without a C-terminal LβH domain, showing diverse forms of oligomerization, from monomers , to octamers ,, (Figures and ).…”

“…Importantly, in most NSPases the LβH domain presents a different orientation with respect to the GT-A like fold, enabling different forms of oligomerization. LβH oligomerization includes (i) top-to-top dimerization as observed in archeal GDP-mannose pyrophosphorylase, (ii) base-to-base dimerization allowing tetramerization as observed in the AGPase from E. coli , (iii) side-by-side parallel trimerization as in the case of GlmU from E. coli , and (iv) top-to-top dimerization allowing octamerization as in eukaryotic UGPases. , …”

Tetramerization is essential for the enzymatic function of the Pseudomonas aeruginosa virulence factor UDP-glucose pyrophosphorylase

“…Therefore, our work aims at identifying unique structural and/or functional features that can be exploited for selective inhibition of the bacterial enzyme. In this regard, mechanistic differences linked to the respective quaternary structure are of specific interest: fungal and animal UGPs form octamers ( 31 – 35 ), and we previously demonstrated that octamerization is required for HsUGP function ( 29 , 36 ). In contrast, plant UGPs are active monomers but appear to be regulated by oligomerization ( 37 – 40 ), whereas protozoan UGPs form exclusively active monomers ( 41 , 42 ).…”