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Genetic and structural validation of Aspergillus fumigatus N-acetylphosphoglucosamine mutase as an antifungal target.

ABSTRACT: Aspergillus fumigatus is the causative agent of IA (invasive aspergillosis) in immunocompromised patients. It possesses a cell wall composed of chitin, glucan and galactomannan, polymeric carbohydrates synthesized by processive glycosyltransferases from intracellular sugar nucleotide donors. Here we demonstrate that A. fumigatus possesses an active AfAGM1 (A. fumigatus N-acetylphosphoglucosamine mutase), a key enzyme in the biosynthesis of UDP (uridine diphosphate)-GlcNAc (N-acetylglucosamine), the nucleotide sugar donor for chitin synthesis. A conditional agm1 mutant revealed the gene to be essential. Reduced expression of agm1 resulted in retarded cell growth and altered cell wall ultrastructure and composition. The crystal structure of AfAGM1 revealed an amino acid change in the active site compared with the human enzyme, which could be exploitable in the design of selective inhibitors. AfAGM1 inhibitors were discovered by high-throughput screening, inhibiting the enzyme with IC50s in the low ?M range. Together, these data provide a platform for the future development of AfAGM1 inhibitors with antifungal activity.

SUBMITTER: Fang W

PROVIDER: S-EPMC3763426 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Genetic and structural validation of Aspergillus fumigatus N-acetylphosphoglucosamine mutase as an antifungal target.

Fang Wenxia W Du Ting T Raimi Olawale G OG Hurtado-Guerrero Ramón R Mariño Karina K Ibrahim Adel F M AF Albarbarawi Osama O Ferguson Michael A J MA Jin Cheng C Van Aalten Daan M F DM

Bioscience reports 20130904 5

Aspergillus fumigatus is the causative agent of IA (invasive aspergillosis) in immunocompromised patients. It possesses a cell wall composed of chitin, glucan and galactomannan, polymeric carbohydrates synthesized by processive glycosyltransferases from intracellular sugar nucleotide donors. Here we demonstrate that A. fumigatus possesses an active AfAGM1 (A. fumigatus N-acetylphosphoglucosamine mutase), a key enzyme in the biosynthesis of UDP (uridine diphosphate)-GlcNAc (N-acetylglucosamine), ...[more]

PMID: 23844980

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Project description:Ergosterol plays an important role in maintaining cell membrane sterol homeostasis in fungi, and as such, it is considered an effective target in antifungal chemotherapy. In yeast, the enzyme acetyl-coenzyme A (CoA) acetyltransferase (ERG10) catalyzes the Claisen condensation of two acetyl-CoA molecules to acetoacetyl-CoA in the ergosterol biosynthesis pathway and is reported as being critical for cell viability. Using yeast ERG10 for alignment, two orthologues, AfERG10A (AFUB_000550) and AfERG10B (AFUB_083570), were discovered in the opportunistic fungal pathogen Aspergillus fumigatus Despite the essentiality of AfERG10B having been previously validated, the biological function of AfERG10A remains unclear. In this study, we have characterized recombinant AfERG10A as a functional acetyl-CoA acetyltransferase catalyzing both synthetic and degradative reactions. Unexpectedly, AfERG10A localizes to the mitochondria in A. fumigatus, as shown by C-terminal green fluorescent protein (GFP) tag fusion. Both knockout and inducible promoter strategies demonstrate that Aferg10A is essential for the survival of A. fumigatus The reduced expression of Aferg10A leads to severe morphological defects and increased susceptibility to oxidative and cell wall stresses. Although the catalytic mechanism of acetyl-CoA acetyltransferase family is highly conserved, the crystal structure of AfERG10A and its complex with CoA are solved, revealing four substitutions within the CoA binding site that are different from human orthologues. Taken together, our combination of genetic and structural studies demonstrates that mitochondrial AfERG10A is essential for A. fumigatus cell viability and could be a potential drug target to feed the antifungal drug development pipeline.IMPORTANCE A growing number of people worldwide are suffering from invasive aspergillosis caused by the human opportunistic fungal pathogen A. fumigatus Current therapeutic options rely on a limited repertoire of antifungals. Ergosterol is an essential component of the fungal cell membrane as well as a target of current antifungals. Approximately 20 enzymes are involved in ergosterol biosynthesis, of which acetyl-CoA acetyltransferase (ACAT) is the first enzyme. Two ACATs in A. fumigatus are AfErg10A and AfErg10B. However, the biological function of AfErg10A is yet to be investigated. In this study, we showed that AfErg10A is localized in the mitochondria and is essential for A. fumigatus survival and morphological development. In combination with structural studies, we validated AfErg10A as a potential drug target that will facilitate the development of novel antifungals and improve the efficiency of existing drugs.

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Dataset Information

Genetic and structural validation of Aspergillus fumigatus N-acetylphosphoglucosamine mutase as an antifungal target.

Publications

Genetic and structural validation of Aspergillus fumigatus N-acetylphosphoglucosamine mutase as an antifungal target.

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