Analysis of NUDIX enzymes across fungi reveals previously unrecognized diversity

“…taxonomic representation of the newly identified subfamilies, we extended the sequence search by using the newly identified fungal NUDIX sequences d as queries in a blastp v. 2.13.0 + search [15] against the NCBI non-redundant (NR) database with an e-value cutoff of 0.001 to collect all homologs.”

“The SAM alignment files were compressed into binary format (BAM) using samtools (v.10) [39].”

“2.5 [32, 33] was performed for the twelve most common NUDIX substrates: 8-oxo-dGDP, 8-oxo-dGTP, ADP-glucose, ADP-ribose, Ap3 A, Ap4 A, Ap6 A, dATP, DHNTP, HSCoA, NAD + and NADPH.”

“19.6) with default settings [37].”

“1.0) [38].”

“The phylogenetic tree was inferred for each of the alignments by maximum likelihood with IQTREE2 [16], considering 1000 bootstrap replicates (parameters: -”

“The redundancy across the identified NUDIX sequences was first reduced with cd-hit (70% sequence identity, 90% coverage, 5 letters word length) [10].”

“The substrates were obtained from the ChEBI database in SMILES format [28] and converted into three-dimensional PDBQT structures with Open Babel 3.1.1 [34] (parameters: --gen3D -p xx -opdbqt ).”

“Trees were visualized and rendered in iToL [18].”

“The phylogenetic tree at the species level was generated with OrthoFinder [17] (parameters: mmseqs and dendroblast) for 41 species spanning the taxonomic diversity of the whole fungal kingdom.”

“To characterize the sequence and structural properties of all fungal NUDIX subfamilies, all of the sequences were scanned against all Pfam family definitions with pfams-can.pl [19] to confirm the presence of the NUDIX domain (PF00293), and look for other coexisting domains.”

“As a reference set of NUDIX enzymes, all human proteins with NUDIX domains were retrieved from the UniProt database (status reviewed) and all NUDIX domain-containing proteins with resolved structures were obtained from PDB [14].”

“Low complexity regions were predicted with the SEG tool [22], coiled-coil regions with EMBOSS pepcoil [23], disordered protein regions with IUPred3 [24], subcellular localization with WoLF PSORT [25], and transmembrane regions were identified with TMHMM [26].”

“To identify NUDIX enzymes across the fungal tree, we first searched all Pfam families in a set of 183 fungal pro-teomes (Supplementary file S1, sheet S1) by amino acid sequence similarity using pfam_scan.pl with default settings against Pfam database v. 36 [9].”

“Each of the subfamilies was represented by two selected sequences and their AlphaFold2 3D structure models [28, 29].”

“Structures were trimmed to the NUDIX domain region in PyMOL and prepared for the molecular docking using pdb2pqr (parameters: --ff AMBER --ph-calc-method = propka --with-ph = xx) [31].”

“Where available, we used NCBI Conserved Domain Database (CDD) [20] annotations for the subfamilies, since CDD enables the identification of conserved domains across multiple protein sequences and defines a more detailed classification of subfamilies of the NUDIX proteins than the Pfam database.”

“The substrates were obtained from the ChEBI database in SMILES format [28] and converted into three-dimensional PDBQT structures with Open Babel 3.1.1 [34] (parameters: --gen3D -p xx -opdbqt ).”

“407 [12].”

“Human Dcp2, NUDT2, NUDT12, and NUDT16 can be used to characterize 5 ′ capped RNA transcripts [1], while OsNUDX14 from Oryza sativa is thought to be a grain quality regulator and to determine plant development, being strongly expressed in mature leaves [5].”

“In agreement with the wide range of functions that they perform, the diversity of the substrates of these enzymes is high, including di-and triphosphates, nucleotide sugars, dinucleosides, diphosphoinositol polyphosphates, and RNA caps [4].”

“Low complexity regions were predicted with the SEG tool [22], coiled-coil regions with EMBOSS pepcoil [23], disordered protein regions with IUPred3 [24], subcellular localization with WoLF PSORT [25], and transmembrane regions were identified with TMHMM [26].”

“Low complexity regions were predicted with the SEG tool [22], coiled-coil regions with EMBOSS pepcoil [23], disordered protein regions with IUPred3 [24], subcellular localization with WoLF PSORT [25], and transmembrane regions were identified with TMHMM [26].”

“All newly identified subfamilies have a well-preserved NUDIX signature motif with at least two glutamates acting as metal ligands and lysine and arginine residues that strengthen catalytic forces [7] (Fig.”

“NUDIX enzymes with feasible druggable sites have also been identified, and therefore they could represent novel drug targets for human diseases, especially in cancer cells [2].”

“ADP-ribosylation is often found as a posttranslational modification of proteins [46].”

“AutoDock Vina predicts the receptor-ligand binding conformations and ranks them according to the predicted change in free energy during binding [35].”

“NUDIX enzymes are ubiquitous in all kingdoms of life and viruses [3, 6, 8], and although these proteins are evolutionarily related, they exhibit significant sequence divergence.”

“NUDIX enzymes are ubiquitous in all kingdoms of life and viruses [3, 6, 8], and although these proteins are evolutionarily related, they exhibit significant sequence divergence.”

“Subfamily F is not present in the available transcriptomes as orchestrators of carotenoid biosynthesis in plants [54].”

“NUDIX enzymes are ubiquitous in all kingdoms of life and viruses [3, 6, 8], and although these proteins are evolutionarily related, they exhibit significant sequence divergence.”

“…are considered to be alar-mones, triggering a stress response that [43] influences a variety of cellular processes such as gene expression and DNA repair [43, 44] Therefore, it is possible that the enzymes of the newly identified subfamilies may modulate the response to cellular stress in fungi.”

“On the other hand, poly-ADP-ribose can accumulate when DNA is damaged [47].”

“Programmed cell death types differ among fungi [45] including apoptosis-like process, heterokaryon incompatibility, and ferroptosis which is a consequence of iron release and lipid peroxidation.”

“Human Dcp2, NUDT2, NUDT12, and NUDT16 can be used to characterize 5 ′ capped RNA transcripts [1], while OsNUDX14 from Oryza sativa is thought to be a grain quality regulator and to determine plant development, being strongly expressed in mature leaves [5].”

“Fungi in this phylum are known to lack several DNA repair and replication genes [48] which could be associated with the abundance of NUDIX enzymes.”

“Although much less is known about fungal pathogens, NUDIX hydrolases have been implicated in the oxidative stress response of Cryptococcus neoformans [53].”

“NUDIX hydrolases are known to be involved in the pathogenicity of a variety of bacteria [51], and there have been attempts to use them as antimicrobial targets [52].”

“NUDIX hydrolases are known to be involved in the pathogenicity of a variety of bacteria [51], and there have been attempts to use them as antimicrobial targets [52].”

“The DIPP subfamily is found across eukaryotes including vertebrates, invertebrates, fungi, and plants but is not found in protists, indicating a later evolution after the divergence of the major domains of life [165,166].”

“The structure of Lng NH exhibits the conserved α/β/α sandwich fold typical of Nudix family proteins (34), comprising five α-helices (α1–α5) and nine β-strands (β1–β9) (Fig.”

“The RNASeq data analysis was carried out based on the steps described by Pasterny et al., 2025 92 .”