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.

Figure 1

Expression and biochemical characterization of Acs1. (A) Reaction scheme of Acs1. (B) Progress curves for detecting the activity of purified recombinant Acs1 for six fungal enzymes using the EnzChek pyrophosphate detection kit (Thermo-Fisher). (C, D) Representative Michaelis–Menten curves for measuring substrate Km’s for CnAcs1; values for all enzymes reported in Table 1.

Figure 2

Overview of fungal orthologs of Acs1 bound to propyl-AMP. Structures shown such that the N-terminal domain (NTD) is light shades, while the C-terminal domain (CTD) and the N-terminal extension (NT-Ext) are in darker shades and the compounds are in spheres. Aspergillus fumigatus (green, PDB 7KDN, 2.8 Å), Candida albicans (blue, PDB 7KDS, 2.9 Å), Coccidioides immitis (pink, PDB 7KQ6, 1.8 Å), Coccidioides posadasii (yellow, PDB 7KCP, 2.15 Å), and Cryptococcus neoformans in two conformations—thioesterification (orange), adenylation (red, PDB 5IFI, 1.95 Å).

Figure 3

Reaction series for Cryptococcus neoformans Acs1. (A) Structure of Cryptococcus neoformans Acs1 (PDB 5PVP, chain A, Apo). (B) Cryptococcus neoformans Acs1 bound to ATP (PDB 5K8F, chain A, AD-conf); (C) bound to acetyl-AMP, adenylating conformation (PDB 74LG, chain B, AD-conf); (D) bound to acetyl-AMP (PDB 74LG, chain C, TE-conf); and (E) bound to propyl-AMP and coenzyme A, thioesterification conformation (PDB 5K85, chain C, TE-conf). Protein model with table for NTD and CTD start residues of other fungal Acs1 proteins. N-terminal domain (NTD, orange), C-terminal domain (CTD, dark red), N-terminal extension (NT-Ext, olive green), hinge (cyan), ATP binding loop (chartreuse), Lys640 in A (blue), potassium (purple), and ligand (yellow).

Figure 4

Bisubstrate inhibitors exhibit submicromolar potency. (A) Structure of the adenosine moiety used for bisubstrate phosphodiester or sulphonamide derivatives. (B, C) Representative CnAcs1 enzyme inhibition kinetics of lead compounds display submicromolar K_i competitive against ATP. (D) SAR of AMP alkyl esters and AMS derivatives for CnAcs1 plotted for potency (K_i) against ATP. Compounds with inhibition above the highest tested concentration are plotted at 50 μM as designated by the horizontal dashed line. No enzyme inhibition was detected by the human ACSS2 inhibitor (VY-3-249).

Figure 5

Thermal shift induced by alkyl AMP esters tightly correlates with inhibitor potency. (A) Thermal profile of fluorescence ratio F350/F330. (B) Thermal profile of the first derivative. The traces in panel A were smoothed using the smoothing function of GraphPad Prism. The T_m of the protein is represented by the peaks in panel B. (C) Correlation of thermal shift from DMSO control with inhibitor potency. (D) Table of inhibitor potency (K_i) and thermal shift (ΔT_m).

Figure 6

Overlay of AMP ester series bound structures forCoccidioides immitis and Cryptococcus neoformans Acs1 crystal structures. (A) Overlay of Coccidioides immitis Acs1 bound to methyl-AMP + coenzyme A (PDB 7L3Q, aquamarine), ethyl-AMP + coenzyme A (PDB 7KVY, teal), and propyl-AMP (PDB 7KQ6, slate). Protein shown in pink. (B) Overlay of Cryptococcus neoformans Acs1 bound to ethyl-AMP (PDB 7KNO, yellow), propyl-AMP + coenzyme A (PDB 5K85, green), and butyl-AMP (PDB 7KNP, blue) in the thioesterification conformation (TE-conf). Protein shown in orange. (C) Overlay of Cryptococcus neoformans Acs1 bound to ethyl-AMP (yellow), propyl-AMP + coenzyme A (green), and butyl-AMP (blue) in the thioesterification conformation (AD-conf). Protein shown in salmon. (D) Overlay of Coccidioides immitis (pink, protein; blue, compound) and Cryptococcus neoformans (protein and compound, orange) propyl-AMP bound crystal structures.

Figure 7

Comparison of Cryptococcus neoformans Acs1 ethyl and butyl-AMP inhibitor bound structures in the AD and TE conformations. (A) Overlay of ethyl-AMP (PDB 7KNO; compound, yellow; protein, pink) and butyl-AMP (PDB 7KNP; compound, blue; protein, dark pink) in the AD-conf. (B) Overlay of ethyl-AMP (PDB 7KNO; compound, yellow; protein, orange) and butyl-AMP (PDB 7KNP, compound, blue; protein, green) in the TE-conf. Red lines indicate new clashes observed.