Structure of Aspergillus fumigatus Cytosolic Thiolase: Trapped Tetrahedral Reaction Intermediates and Activation by Monovalent Cations

Abstract

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Engineering potassium activation into biosynthetic thiolase

“…Here and elsewhere [16], we have compared ACAT X-ray crystal structures and show that very few major structural features differentiate each subtype, leading us to hypothesise that K + -activation may be encoded by only a small number of residues. By assaying a range of variant enzymes in vitro, here we show that K + -activation can be engineered into bCT by substituting only two amino acids.…”

“…This is the first step to produce a carbon skeletal framework for a myriad of important biomolecules such as energy-and carbon-storage polyesters in many bacteria [13], and isoprenoids in eukaryotes, which include sterols, dolichols and many antioxidants, pigments and pharmaceuticals [14,15]. The thiolase catalytic cycle involves two acetyl-transfer reactions: one from acetyl-CoA to a nucleophilic cysteine residue at the active site, Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20210455/918155/bcj-2021-0455.pdf by guest on 09 August 2021 forming an acetylated enzyme intermediate, followed by a second acetyl-transfer to a second acetyl-CoA molecule to produce acetoacetyl-CoA [16]. Cytosolic thiolase from the bacterium, Zoogloea ramigera (referred to herein as bacterial cytosolic thiolase; bCT) has been studied in detail, highlighting all of the critical catalytic residues [17,18].…”

“…X-ray crystal structures of bCT in apo, acetylated, substrate-bound and product-bound forms have been solved previously [19][20][21]. Recently, we have solved crystal structures of cytosolic thiolase from the filamentous fungus, Aspergillus fumigatus (afCT), in complex with each tetrahedral reaction intermediate that occurs during each of the two acetyl-transfer reactions, completing a full structural repertoire of every step in the thiolase catalytic cycle [16].…”

“…This led to the assumption that K + -activation was peculiar to mitochondrial ACATs, and forms the basis of a common test for diagnosing β-ketothiolase deficiency [22]. The recent demonstration that afCTa fungal cytosolic ACATis also K + -activated has challenged this paradigm and suggested more complex evolutionary paths to each subtype [16]. The existence of M + -activated and M + -independent enzymes in the same conserved family presents opportunities for structural studies to understand the mechanism of allosteric M + -activation.…”

Targeting Unconventional Pathways in Pursuit of Novel Antifungals

“…Increases in disease incidence and prevalence of antifungal resistance highlights the need to identify novel targets and develop new classes of antifungals to manage mycoses amongst the immunocompromised population. There have been continual efforts to characterize enzymes involved in the biosynthesis of ergosterol or cell wall components, both of which are classic antifungal targets, to develop novel inhibitors (Urbina et al, 2000 ; Hata et al, 2010 ; Marshall et al, 2018 ). However, there has also been a notable shift in focus from these pathways exclusive to fungi to exploiting species-specific differences in shared pathways between fungi and humans (Rodriguez-Suarez et al, 2007 ; Marshall et al, 2017 , 2019 ; Kummari et al, 2018 ).…”