Identification and Characterization of Two Aryl Sulfotransferases from Deep-Sea Marine Fungi and Their Implications in the Sulfation of Secondary Metabolites

Abstract

Sulfation plays a critical role in the biosynthesis of small molecules, regulatory mechanisms such as hormone signaling, and detoxification processes (phase II enzymes). The sulfation reaction is catalyzed by a broad family of enzymes known as sulfotransferases (SULTs), which have been extensively studied in animals due to their medical importance, but also in plant key processes. Despite the identification of some sulfated metabolites in fungi, the mechanisms underlying fungal sulfation remain largely unknown. To address this knowledge gap, we conducted a comprehensive search of available genomes, resulting in the identification of 174 putative SULT genes in the Ascomycota phylum. Phylogenetic analysis and structural modeling revealed that these SULTs belong to the aryl sulfotransferase family, and they are divided into two potential distinct clusters of PAPS-dependent SULTs within the fungal kingdom. SULT genes from two marine fungi isolated from deep-sea hydrothermal vents, Hortaea werneckii UBOCC-A-208029 (HwSULT) and Aspergillus sydowii UBOCC-A-108050 SULT (AsSULT), were selected as representatives of each cluster. Recombinant proteins were expressed in Escherichia coli and biochemically characterized. HwSULT demonstrated high and versatile activity, while AsSULT appeared more substrate-specific. Here, HwSULT was used to sulfate the mycotoxin zearalenone, enhancing its cytotoxicity toward healthy feline intestinal cells.

Keywords: cytotoxic activity; enzymatic sulfation; marine fungi; sulfated metabolites; sulfated mycotoxin; sulfotransferases.

Figure 1

General mechanism of PAPS-dependent sulfotransferase.

Figure 2

Phylogenetic tree of putative fungal SULTs. Sulfotransferase domains were aligned and phylogeny analyzed by maximum likelihood (RaxMl v. 8.2.12). The reliability of the trees was tested by bootstrap analysis using 100 resamplings of the dataset. Only bootstrap values above 50% are shown. Fungi are colored in dark blue, humans in black, and A. thaliana in green. Accession numbers of used proteins are listed in Table S1.

Figure 3

PF00685/St1-1 aryl sulfotransferase (SULT) family phylogenetic tree. Sulfotransferase domains were aligned and manually curated, resulting in 160 informative positions. Phylogeny was analyzed by maximum likelihood (RaxMl v. 8.2.4). The reliability of the trees was tested by bootstrap analysis using 100 resamplings of the dataset. Only bootstrap values above 50% are shown. Fungi are colored in dark blue. Accession numbers of used proteins are listed in Table S2.

Figure 4

Putative PAPS-binding sites (5′PSB and 3′PB) and catalytic histidine in AsSULT and HwSULT. Partial alignment was performed with human SULT1B1 (PDB: 3CKL), SULT1C2 (PDB: 3BFX), SULT2A1 (PDB: 4IFB), SULT2B1 (PDB: 1Q22), and Arabidopsis thaliana SOT16 (PDB: 8K9Y) and SOT18 (PDB: 5MEX), whose structures were solved in the presence of PAPS. Two prokaryotic SULTs were added, StaL (PDB: 2OV8) from Streptomyces toyocaensis NRRL 15009 and the biochemically characterized SULT from Mycobacterium tuberculosis H37Rv^T (UniProt: P9WGB9). Consensus threshold 70% (bolded residues). Secondary structures derived from PDB files or predicted by AlphaFold2 (AF2) for HwSULT, FgSULT1, AsSULT, and Glycolipid sulfotransferase. Blue boxes indicate residues involved in hydrogen or ionic bonds with PAPS. Orange boxes indicate residues involved in aromatic–aromatic interaction with adenine moiety of PAPS. Cyan boxes represent catalytic histidine.

Figure 5

Conservation of 5′PSB, catalytic histidine, and 3′PB among fungal SULTs. The frequency plot is a graphical representation of a multiple sequence alignment of the dataset of 174 sequences. The letter height is proportional to the conservation of the residue.

Figure 6

Conservation of the PAPS-binding pocket in HwSULT and AsSULT. (A) Overimposition of PAPS-binding residues in ST1B1 (PDB: 3CKL), HwSULT, and AsSULT. (B) PAPS-binding residues in SULT1B1 (PDB: 3CKL). (C) Inferred PAPS-binding residues in HwSULT. (D) Inferred PAPS-binding residues in AsSULT. (E) Alignment of PAPS-binding residues. Green boxes indicate conserved residues. Orange boxes indicate partially conserved residues. Red boxes indicate different residues.

Figure 7

Modeling of putative entrances and tunnels leading to the PAPS-binding pocket in the SULT1B1 structure (PDB: 3CKL), HwSULT, and AsSULT models. CaveR webtool 1.2 was used to calculate entries and tunnels.

Figure 8

Conservation of a dimerization motif in AsSULT and in proteins belonging to the Aspergillus cluster. (A) Putative dimerization motif (KxxxTVxxxE) in AsSULT. Consensus threshold 70% (bolded residues). Green boxes indicate residues involved in dimerization. (B) AsSULT homodimer best model overimposed on SULT2A1 homodimer. (C) Conservation of the dimerization motif (KxxxTVxxxE) among fungal SULTs. The frequency plot is a graphical representation of a multiple sequence alignment of the dataset of 174 sequences. The letter height is proportional to the conservation of the residue.

Figure 9

Graphical representation of substrates sulfated by HwSULT and AsSULT detected by LC-MS. R_1-2-3 correspond to the putative sulfate position, R_1-2-3 = H or SO₃. The color of each chromatogram corresponds to the color of the associated chemical structure.

Figure 10

Impact of sulfation on the bioactivity of (A) phloroglucinol (PGL) and (B) zearalenone (ZEA) in feline intestinal cells (n = 9). One-way ANOVA results are represented by different letters, which indicate significant differences at p < 0.05.