A characterization of a large subset of PAAT enzymes, including their likely oligomeric states, functional site properties, and experimental functions is presented, including their likely oligomeric states, functional site properties, and experimental functions.
Abstract
Gcn5-related N-acetyltransferases (GNATs) are considered a "megafamily" that ranks among the most structurally and sequentially diverse superfamilies in the CATH (Class, Architecture, Topology, Homology) database. In this vast superfamily, several types of protein functions have been explored throughout evolution, yet the evolutionary pathways that led to such diversity remain poorly understood. To investigate these concepts further, we selected a functionally distinct GNAT subgroup called polyamine N-acetyltransferases (PAATs). These enzymes acetylate polyamines that are crucial for cellular homeostasis. While PAATs from different domains of life catalyze the same reaction, their residue conservation patterns, oligomeric states, and presence of allosteric sites vary. Despite their biological importance, many putative PAATs remain uncharacterized, limiting our ability to infer evolutionary relationships, understand how functional properties emerged, and appreciate the extent of their structural diversity and substrate specificity. Here, we present a characterization of a large subset of PAAT enzymes, including their likely oligomeric states, functional site properties, and experimental functions.
The analysis resolves an expanded BECR-fold landscape comprising canonical BECR and BECR-like superfamilies, refines the organization of canonical BECR proteins and identifies previously unrecognized families, and expands and clarify the BECR-fold landscape.
Kristi Pham, Gianlucca G. Nicastro, Audrey R. Long et al.· bioRxiv· 0 citations
These findings reconstruct the evolutionary emergence of the DGF-1 architecture from pre-existing structural modules and provide a framework for one of the largest and most enigmatic gene families in trypanosomatids.
Mathias J. Mangino, Juan Manuel Trinidad-Barnech, A. Parodi-Talice et al.· bioRxiv· 0 citations
The RNase-H fold is an ancient protein fold found in diverse nucleases that is characterised by a conserved structural core (five β strands and ⍺ helices), canonical DDE/D catalytic residues and catalytic mechanism. This study focuses on the evolutionary conservation of the RNaseH-like domain in the transposase-derived THAP9 family among different taxonomic groups. Phylogenetic analysis demonstrates that the structural framework required for catalysis has been maintained over the course of evolution, while other peripheral regions have undergone lineage-specific diversification. This is exemplified by a Lys residue, which is conserved across mammals, can be structurally superposed on a catalytic Asp residue found in active transposases, but is not important for DNA integration. Further, the importance of conserved motifs (YREK and YKEFR motifs) near the THAP9 catalytic residues are confirmed biochemically by performing site directed mutagenesis followed by functional assays which demonstrate that substitution mutants have decreased ability to integrate DNA. Overall, these results emphasize that DNA integration activity is controlled by interactions with several structural elements rather than by individual catalytic residues.
Serine racemase (SR) catalyzes the interconversion of l-serine and d-serine and can also catalyze β-elimination of serine to pyruvate and ammonia. In mammals, however, the presence and enzymatic function of SR have been reported mainly in mouse, rat, and human. It is therefore unclear whether functional SR is broadly conserved across mammals or whether its enzymatic properties differ among lineages. To address this question, we produced and purified eleven SRs from representative eutherians, marsupials, and a monotreme and compared their catalytic properties. All enzymes examined showed both racemization and β-elimination activities. Together with the phylogenetic relationships, these activities suggest that functional SR was present before the monotreme-therian divergence and retained in the lineages examined. Specific activities and kinetic parameters nevertheless differed among species. Residues involved in cofactor and substrate binding were broadly conserved, whereas variation at the substrate-recognition region around positions 150-152 may contribute to the observed kinetic differences. Analysis of public transcriptome data further suggested species differences in tissue expression patterns. Together, these findings show conservation of SR function across the mammalian lineages examined, with species-dependent differences in catalytic properties and expression.
K. Uda, Luke A. Moe· Comparative Biochemistry and...· 0 citations
Deoxyguanosine triphosphatases (dGTPases) are nucleotide-depleting enzymes known to play a role in antiviral defense. While their enzymatic mechanism is established, the structural and functional diversity of dGTPases remains poorly understood. Here, we report a systematic analysis of dGTPase homologs across bacteria, archaea, and eukaryotes, revealing their widespread distribution and association with diverse immune-related domains. Through integrative bioinformatics and structural mining, we identify a class of bacterial dGTPases that assemble into stable octameric and higher-order oligomeric structures. Using cryo-electron microscopy, we resolve the octameric and 16-mer assemblies of a representative Vibrio dGTPase (Vdg) and further captured filamentous forms. Functional assays demonstrate that octamer formation is essential and sufficient for antiviral activity, while higher-order assemblies are dispensable. We also identify dAMP as an allosteric regulator, underscoring the functional versatility of dGTPases. Our findings provide insights into the modular architecture, oligomerization-driven activation, and immune function of bacterial dGTPases, and broaden our understanding of nucleotide depletion-based antiviral strategies. Deoxyguanosine triphosphatases (dGTPases) are nucleotide-depleting enzymes that contribute to antiviral defense. Here, the authors reveal widespread and structurally diverse dGTPases and characterize oligomeric dGTPases in distinct assembly states, providing insights into their architecture, activation, and regulation.
Miao Shi, Peipei Li, Quanjin Li et al.· Nature Communications· 0 citations
Nucleases are crucial for various bacterial processes, including genome maintenance and host defense. Deoxydinucleases (diDNases), a class of Gram-positive bacteria–specific nucleases associated with mobile genetic elements, are homologous to nanoRNase C (NrnC) in Gram-negative bacteria but exhibit notable differences: diDNases form dimers and cleave DNA dinucleotides, whereas NrnC forms octamers that process both RNA and DNA dinucleotides. The mechanism by which substrate specificity emerged, and whether it is linked to oligomerization, remained unknown. Here, we reconstructed a common ancestor of diDNases and NrnC orthologs that forms a dimer with intermediate preference for DNA. Structures of ancestral and extant dinucleases reveal gradual changes in conformation that gave rise to substrate preference, oligomeric state, and catalytic efficiency. These findings highlight how subtle, concerted structural modifications enable large-scale changes in molecular assembly and functional specialization, harnessing a conserved protein fold. DNA dinucleotide preference in the early ancestor and preservation of DNase activity in all extant enzymes strongly argue for a biological function of DNA dinucleotides.
Sofia Mortensen, Audrey A. Burnim, K. Dufault-Thompson et al.· Science Advances· 0 citations
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