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Structural Characterization of the Reaction and Substrate Specificity Mechanisms of Pathogenic Fungal Acetyl-CoA Synthetases.

ABSTRACT: Acetyl CoA synthetases (ACSs) are Acyl-CoA/NRPS/Luciferase (ANL) superfamily enzymes that couple acetate with CoA to generate acetyl CoA, a key component of central carbon metabolism in eukaryotes and prokaryotes. Normal mammalian cells are not dependent on ACSs, while tumor cells, fungi, and parasites rely on acetate as a precursor for acetyl CoA. Consequently, ACSs have emerged as a potential drug target. As part of a program to develop antifungal ACS inhibitors, we characterized fungal ACSs from five diverse human fungal pathogens using biochemical and structural studies. ACSs catalyze a two-step reaction involving adenylation of acetate followed by thioesterification with CoA. Our structural studies captured each step of these two half-reactions including the acetyl-adenylate intermediate of the first half-reaction in both the adenylation conformation and the thioesterification conformation and thus provide a detailed picture of the reaction mechanism. We also used a systematic series of increasingly larger alkyl adenosine esters as chemical probes to characterize the structural basis of the exquisite ACS specificity for acetate over larger carboxylic acid substrates. Consistent with previous biochemical and genetic data for other enzymes, structures of fungal ACSs with these probes bound show that a key tryptophan residue limits the size of the alkyl binding site and forces larger alkyl chains to adopt high energy conformers, disfavoring their efficient binding. Together, our analysis provides highly detailed structural models for both the reaction mechanism and substrate specificity that should be useful in designing selective inhibitors of eukaryotic ACSs as potential anticancer, antifungal, and antiparasitic drugs.

SUBMITTER: Jezewski AJ

PROVIDER: S-EPMC8383264 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Cytosolic acetyl-coenzyme A is a precursor for many biotechnologically relevant compounds produced by Saccharomyces cerevisiae. In this yeast, cytosolic acetyl-CoA synthesis and growth strictly depend on expression of either the Acs1 or Acs2 isoenzyme of acetyl-CoA synthetase (ACS). Since hydrolysis of ATP to AMP and pyrophosphate in the ACS reaction constrains maximum yields of acetyl-CoA-derived products, this study explores replacement of ACS by two ATP-independent pathways for acetyl-CoA synthesis. After evaluating expression of different bacterial genes encoding acetylating acetaldehyde dehydrogenase (A-ALD) and pyruvate-formate lyase (PFL), acs1M-NM-^T acs2M-NM-^T S. cerevisiae strains were constructed in which A-ALD or PFL successfully replaced ACS. In A-ALD-dependent strains, aerobic growth rates of up to 0.27 h-1 were observed, while anaerobic growth rates of PFL-dependent S. cerevisiae (0.21 h-1) were stoichiometrically coupled to formate production. In glucose-limited chemostat cultures, intracellular metabolite analysis did not reveal major differences between A-ALD-dependent and reference strains. However, biomass yields on glucose of A-ALD- and PFL-dependent strains were lower than those of the reference strain. Transcriptome analysis suggested that reduced biomass yields were caused by acetaldehyde and formate in A-ALD- and PFL-dependent strains, respectively. Transcript profiles also indicated that a previously proposed role of Acs2 in histone acetylation is probably linked to cytosolic acetyl-CoA levels rather than to direct involvement of Acs2 in histone acetylation. While, for the first time, demonstrating that yeast ACS can be fully replaced by alternative reactions, this study demonstrates that further modifications are needed to achieve optimal in vivo efficiencies of the supply of acetyl-CoA as product precursor. To investigate the impact of introduced changes in native pathway of cytosolic acetyl-CoA formation in S. cerevisiae, a DNA microarray-based transcriptome analysis was performed on aerobic or anaerobic, glucose-limited chemostat cultures.

Project description:BACKGROUND: Most filamentous ascomycete fungi produce high affinity iron chelators called siderophores, biosynthesized nonribosomally by multimodular adenylating enzymes called nonribosomal peptide synthetases (NRPSs). While genes encoding the majority of NRPSs are intermittently distributed across the fungal kingdom, those encoding ferrichrome synthetase NRPSs, responsible for biosynthesis of ferrichrome siderophores, are conserved, which offers an opportunity to trace their evolution and the genesis of their multimodular domain architecture. Furthermore, since the chemistry of many ferrichromes is known, the biochemical and structural 'rules' guiding NRPS substrate choice can be addressed using protein structural modeling and evolutionary approaches. RESULTS: A search of forty-nine complete fungal genome sequences revealed that, with the exception of Schizosaccharomyces pombe, none of the yeast, chytrid, or zygomycete genomes contained a candidate ferrichrome synthetase. In contrast, all filamentous ascomycetes queried contained at least one, while presence and numbers in basidiomycetes varied. Genes encoding ferrichrome synthetases were monophyletic when analyzed with other NRPSs. Phylogenetic analyses provided support for an ancestral duplication event resulting in two main lineages. They also supported the proposed hypothesis that ferrichrome synthetases derive from an ancestral hexamodular gene, likely created by tandem duplication of complete NRPS modules. Recurrent losses of individual domains or complete modules from this ancestral gene best explain the diversity of extant domain architectures observed. Key residues and regions in the adenylation domain pocket involved in substrate choice and for binding the amino and carboxy termini of the substrate were identified. CONCLUSION: Iron-chelating ferrichrome synthetases appear restricted to fission yeast, filamentous ascomycetes, and basidiomycetes and fall into two main lineages. Phylogenetic analyses suggest that loss of domains or modules led to evolution of iterative biosynthetic mechanisms that allow flexibility in biosynthesis of the ferrichrome product. The 10 amino acid NRPS code, proposed earlier, failed when we tried to infer substrate preference. Instead, our analyses point to several regions of the binding pocket important in substrate choice and suggest that two positions of the code are involved in substrate anchoring, not substrate choice.

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Structural Characterization of the Reaction and Substrate Specificity Mechanisms of Pathogenic Fungal Acetyl-CoA Synthetases.

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