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

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.


 EMENI for Aspergillus nidulans strain FGSC A4 (teleomorph Emericella nidulans)
It is apparent that UniProt Taxonomy provides taxon mnemonics that are derived from the teleomorph names of F. verticillioides, F. graminearum and A. nidulans. As this is an unavoidable limitation of nomenclature, we try to facilitate the reader of this manuscript by providing both the anamorph and teleomorph names of each fungus in the beginning of the Results and Methods sections, as well as in Table 1 and in all figure legends. We link this nomenclature to the official taxon mnemonics attached to the symbols of NAT homologues.  and used the same chromatographic fraction (typically from elution with 50 mM imidazol) of the same protein preparation per set of enzymatic activity assays.

Enzyme activity assays with recombinant proteins
Each 50 μl reaction was initiated by addition of 0.4 mM acyl-CoA to purified recombinant NAT protein (0.5 or 1 μg) and 0.5 mM of substrate in 20 mM Tris-HCl (pH 7.5) buffer. Reactions were terminated after 0-15 min, by addition of 12.5 μl of reagent containing 5 mM 5,5′-dithiobis-2-nitrobenzoic acid (Ellman's reagent) and 6.4 M guanidine-HCl in 0.1 M Tris-HCl (pH 7.5). The absorbance of the coloured product was quicky measured at 405 nm using a TECAN M1000 microplate reader.
Assays were performed in duplicate and control reactions, lacking recombinant NAT protein, where performed to assess possible spontaneous hydrolysis of acyl-CoA compounds during the reaction. Endogenous NAT activity in E. coli is below detectable levels when affinity chromatography purified preparations of recombinant proteins are used in assays. The amount (nmol) of produced CoA was determined from linear (R 2 >99.5%) plots of OD (405 nm) against a series of standards containing 0-17.5 nmol of CoA and 0.5 mM substrate. Enzyme specific activity was determined as nmol of produced CoA per min per mg of recombinant protein.

Enzyme activity assays with cell extracts
Enzyme activity assays were performed in 100 μl reactions containing 80-110 μg of total soluble protein, 0.1 mM of 3,4DCA and 0.4 mM of acetyl-CoA or malonyl-CoA in 20 mM Tris-HCl (pH 7.5), 1 mM dithiothreitol (added fresh). After specific incubation times (typically 0-12 min, or up to 30 min), reactions were terminated with 20% w/v trichloroacetic acid. Following addition of 5% w/v p-dimethylaminobenzaldehyde in 9:1 v/v acetonitrile:water (Erlich's reagent), absorbance was measured at 450 nm using a conventional Beckman Coulter DU 730 spectrophotometer. All reactions were performed in triplicate and a no lysate control was always included to assess the inherent stability of the arylamine under the assay conditions applied (for example, 3,4DCA was preferred over 2AP, as it was determined to be considerably more stable during prolonged incubation periods). The amount (nmol) of substrate was determined from linear (R 2 >99.9%) plots of OD (450 nm) against a series of 3,4DCA standards (0-30 nmol). Enzyme specific activity was determined as nmol of consumed arylamine per min per mg of total protein.

Acceptor substrates used in enzyme assays
The following arylamines and arylhydrazines were used in the study (abbreviations