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- Steroids are found in bacteria and eukaryotes, and genes potentially encoding steroid metabolic enzymes have also been identified in giant viruses. For decades, hydroxylated steroids have been utilized in medicine to treat various human diseases. The hydroxylation of steroids can be achieved using microbial enzymes, especially cytochrome P450 monooxygenases (CYPs/P450s) and is well documented. Understanding the structural determinants that govern the regio- and stereoselectivity of steroid hydroxylation by P450s is essential in order to fully exploit their potential. Herein, we present a comprehensive analysis of the steroid-hydroxylating CYP109 family across the domains of life and delineate the structural determinants that govern steroid hydroxylation. Data mining, annotation, and phylogenetic analysis revealed that CYP109 family members are highly populated in bacteria, and indeed, these members passed from bacteria to archaea by horizontal gene transfer, leading to the evolution of P450s in archaea. Analysis of twelve CYP109 crystal structures revealed large, flexible, and dynamic active site cavities that can accommodate multiple ligands. The correct positioning and orientation of the steroid in the active site cavity and the nature of the C17 substituent on the steroid molecule influence catalysis. In an analogous fashion to the CYP107 family, the amino acid residues within the CYP109 binding pocket involve hydrophilic and hydrophobic interactions, influencing substrate orientations and anchoring and determining the site of hydroxylation and catalytic activity. A handful of amino acids, such as Val84, Val292, and Ser387 in CYP109B4, have been found to play a role in determining the catalytic regiospecificity, and a single amino acid, such as Arg74 in CYP109A2, has been found to be essential for the enzymatic activity. This work serves as a reference for the precise understanding of CYP109 structure–function relationships and for P450 enzymes in general. The findings will guide the genetic engineering of CYP109 enzymes to produce valuable steroid molecules of medicinal and biotechnological importance.
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- 2025| Multidisciplinary Di...Proline-, Glutamic acid-, Leucine-rich Protein 1 (PELP1) is a multifunctional nuclear protein essential for ribosome biogenesis and steroid receptor signaling. It contains two hallmark domains: the RIX1 (Ribosome Export 1) domain, which mediates rRNA processing, and the NUC (nucleolar) domain, associated with nucleolar function. While PELP1’s biological roles are well-characterized in mammals, particularly Homo sapiens, its distribution, structural diversity, and evolutionary origin across the domain of life remain largely unexplored. This study addresses this gap by conducting a comprehensive data mining of PELP1 proteins across the NCBI, UniProt, and EukProt databases. A total of 646 PELP1 proteins were identified exclusively in eukaryotes, specifically within the Opisthokonta clade, comprising Fungi, Filasterea, and Metazoa, while no homologs were detected in Bacteria, Viruses, Plants, or Oomycota. Domain analysis revealed that PELP1 proteins contain one RIX1 domain and one or two NUC202 domains. Motif analysis identified LXXLL and PXXP motifs, indicative of receptor-mediated signaling capability, although leucine and proline residues were not universally conserved within these motifs. Amino acid composition analysis showed enrichment of proline, glutamic acid, and cysteine across most PELP1 proteins. Despite low overall sequence identity, structural modeling demonstrated strong conservation of the α-helical fold, with an average root-mean-square deviation (RMSD) of 1.9 Å across species. Evolutionary analysis suggests that ancestral PELP1 emerged before the divergence of opisthokonts, originating from an RIX1-domain-containing protein that subsequently acquired a NUC202 domain. Phylogenetic clustering and sequence identity patterns resolved three major evolutionary lineages corresponding to fungi, filastereans, and metazoans. Overall, these findings reveal that PELP1 proteins exhibit extensive sequence divergence while maintaining a conserved structural architecture, reflecting evolutionary adaptation that preserves functional integrity across opisthokonts.
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