Homologues of xenobiotic metabolizing N-acetyltransferases in plant-associated fungi: Novel functions for an old enzyme family

Abstract

Plant-pathogenic fungi and their hosts engage in chemical warfare, attacking each other with toxic products of secondary metabolism and defending themselves via an arsenal of xenobiotic metabolizing enzymes. One such enzyme is homologous to arylamine N-acetyltransferase (NAT) and has been identified in Fusarium infecting cereal plants as responsible for detoxification of host defence compound 2-benzoxazolinone. Here we investigate functional diversification of NAT enzymes in crop-compromising species of Fusarium and Aspergillus, identifying three groups of homologues: Isoenzymes of the first group are found in all species and catalyse reactions with acetyl-CoA or propionyl-CoA. The second group is restricted to the plant pathogens and is active with malonyl-CoA in Fusarium species infecting cereals. The third group generates minimal activity with acyl-CoA compounds that bind non-selectively to the proteins. We propose that fungal NAT isoenzymes may have evolved to perform diverse functions, potentially relevant to pathogen fitness, acetyl-CoA/propionyl-CoA intracellular balance and secondary metabolism.

Figure 1. Activity of fungal NAT enzymes with different acyl-coenzyme A compounds.

Overview of the acyl-CoA selectivity pattern observed for the recombinant NAT isoenzymes of F. verticillioides (G. moniliformis-GIBM7), F. graminearum (G. zeae-GIBZE), F. oxysporum f.sp. lycopersici (FUSO4), A. flavus (ASPFN) and A. nidulans (E. nidulans-EMENI). Functional homologues are grouped together within coloured boxes labelled I-III. Each enzyme was assayed against a series of acyl-CoA compounds, used as acyl-group donors in reactions with 5-aminosalicylate as acceptor substrate. The results for each set of assays are presented in Supplementary Fig. S3.

Figure 2. Effect of acyl-coenzyme A compounds on the Tm of fungal NAT proteins.

Overview of Tm values determined by differential scanning fluorimetry for recombinant NAT isoenzymes of F. verticillioides (G. moniliformis-GIBM7), F. graminearum (G. zeae-GIBZE), F. oxysporum f.sp. lycopersici (FUSO4) and A. flavus (ASPFN), in the absence or presence of various acyl-CoAs. Two replicate experiments were performed, generating overlapping curves for which the average Tm (± standard deviation) is shown. The results for each set of experiments are presented in Supplementary Fig. S4.

Figure 3. Activity of fungal NAT enzymes with different acceptor substrates.

Overview of the acceptor substrate selectivity pattern observed for group I and II isoenzymes of F. verticillioides (G. moniliformis-GIBM7), F. graminearum (G. zeae-GIBZE), F. oxysporum f.sp. lycopersici (FUSO4), A. flavus (ASPFN) and A. nidulans (E. nidulans-EMENI). Recombinant NAT proteins were assayed with selective acyl-CoAs against a panel of arylamine and arylhydrazine substrates, and the results for each set of assays are presented in Supplementary Fig. S5. The full chemical names of compounds are: 2-aminophenol (2AP), 4-aminoveratrole (AMV), 4-anisidine (ANS), 4-chloroaniline (CLA), 3,4-dichloroaniline (3,4DCA), 4-phenoxyaniline (POA), hydralazine (HDZ), 4-aminosalicylate (4AS), 4-aminobenzoate (PABA), isoniazid (INH), procainamide (PA) and sulphamethazine (SMZ).

Figure 4. NAT enzymatic activities in fungal cell extracts upon xenobiotic exposure.

Erlich’s reagent was used to measure NAT activity in fungal soluble extracts, following enzyme assays with 3,4-dichloroaniline (3,4DCA) and either acetyl- or malonyl-CoA. The graphs show comparison of NAT enzymatic activities measured in cell extracts from cultures challenged for 2 h with xenobiotics (mixture of 2-benzoxazolinone and 3,4DCA, each at 25 μg/ml), relative to extracts prepared from cultures grown in standard medium. Control assays, without cell extract, are also shown. Each data point is the average value of three replicates ± standard deviation. Results are shown for assays performed with cell extracts from F. verticillioides (G. moniliformis) strain FGSC 7600 (a), F. graminearum (G. zeae) strain PH-1 (b), F. oxysporum f.sp. lycopersici strain FOL 4287 (c) and A. flavus strain NRRL 3357 (d). The effects of xenobiotics on NAT enzymatic activity measured with malonyl-CoA in cell extracts from F. verticillioides strains FGSC 7600, MRC 826 and JFL A00999 are also compared (e).

Figure 5. Fungal tolerance of 2-benzoxazolinone.

F. verticillioides (G. moniliformis) strain FGSC 7600 (a), F. graminearum (G. zeae) strain PH-1 (b), F. oxysporum f.sp. lycopersici strain FOL 4287 (c), A. flavus strain NRRL 3357 (d) and A. nidulans (E. nidulans) strain FGSC A4 (e) were grown on standard agar medium supplemented with up to 1000 μg/ml of 2-benzoxazolinone. Cultures are shown after 5 days of incubation.