Unveiling the Evolutionary, Structural, and Regulatory Insights of a Novel Urocanate Hydratase in Acinetobacter sp. Strain DF4

doi:10.21203/rs.3.rs-4360723/v1

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Unveiling the Evolutionary, Structural, and Regulatory Insights of a Novel Urocanate Hydratase in Acinetobacter sp. Strain DF4

https://doi.org/10.21203/rs.3.rs-4360723/v1

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This study delves into the characterization of a novel urocanate hydratase enzyme identified in Acinetobacter sp. strain DF4, emphasizing its taxonomic distribution, conservation patterns, and functional implications. The investigation revealed a skewed taxonomic distribution of ORF1 homologs primarily within the Pseudomonadota phylum, highlighting its deeply conserved function, particularly in Gammaproteobacteria and Acinetobacter species. Homology analyses confirmed close relationships to known urocanate hydratases across bacterial species, reinforcing its role in histidine catabolism pathways. Structural analyses revealed distinct sub-domains within ORF1, suggesting potential NAD binding sites and functional roles. Conservation patterns of the NWCEFD and NWEHFN motifs across diverse organisms underscored their evolutionary significance and potential functional conservation. Post-translational modification predictions indicated multiple phosphorylation and N-myristoylation sites that may impact protein function. Transcriptional regulation elements identified in the ORF1 sequence suggested a complex regulatory network, with putative binding sites for various transcription factors and elements essential for translation initiation and RNA polymerase binding. These findings collectively provide valuable insights into the evolutionary relationships, structural features, and regulatory mechanisms governing the novel urocanate hydratase enzyme in Acinetobacter sp. strain DF4.

Urocanase hydratase

Acientobacter

Taxonomic Distribution

Conservation Patterns

Post-Translational Modifications

Transcriptional Regulation

Bacteria, ubiquitous inhabitants of our planet, had evolved a remarkable diversity of metabolic pathways. This adaptation enabled them to thrive in a multitude of ecological niches by efficiently exploiting various resources [1]. Unveiling these intricate metabolic landscapes proved paramount for several reasons. It provided a window into bacterial physiology and adaptation strategies, elucidating how they navigated the challenges posed by diverse environments [2]. Furthermore, it offered new avenues to combat the growing threat of antibiotic resistance. By understanding these pathways, scientists were able to develop novel antibiotics that targeted specific bacterial metabolic processes, providing potential solutions to this critical public health challenge [3].

Histidine catabolism emerged as a critical pathway within the intricate network of bacterial metabolism. This essential amino acid played a multifaceted role in bacterial life, influencing fundamental processes like growth, virulence, and stress response [4]. Deciphering the enzymes involved in histidine catabolism across various bacterial strains became essential for understanding the interplay between metabolic networks. This knowledge paved the way for targeted strategies to combat pathogenic bacteria by disrupting these critical pathways [5].

The present research delved into the intriguing world of a previously uncharacterized urocanate hydratase enzyme found in Acinetobacter sp. strain DF4 [6]. This unique enzyme represented a missing piece in the puzzle of histidine catabolism within this specific bacterial strain. By employing a multifaceted approach that integrated bioinformatics analysis, homology modeling, and ligand binding assays, the research aimed to elucidate the structure-function relationship of this novel enzyme. Through this endeavor, it not only contributed to a deeper understanding of Acinetobacter sp.'s metabolic machinery but also shed light on the broader landscape of urocanate hydratase diversity and functions within the bacterial kingdom.

The subsequent sections of this paper would delve into the meticulous processes involved in characterizing this intriguing enzyme. It would detail its cloning, sequencing, and functional validation, unveiling the secrets it held. The belief was that these findings would not only contribute to a deeper understanding of bacterial metabolism but also highlight the potential impact of such research on various scientific disciplines, from medicine to biotechnology.

Cloning and Sequencing

A bioluminescent bioreporter for phenol was constructed using the mopR-like promoter segment from strain DF4 [6]. Specific primers targeted regions of the mopR sequence for amplifying the 0.72-kb promoter segment, which was then cloned into the pCR2.1-TOPO vector. Following digestion with NotI and XbaI, the 0.72-kb fragment was ligated into a lux cassette from Vibrio fischeri on the pUTK215 vector. The resulting construct, pUTK258, was confirmed by sequencing post-transformation into Escherichia coli SV17. Subsequent mating of E. coli SV17 pUTK258 with Acinetobacter sp. strain DF4 led to colony selection based on light production in the presence of phenol, with DF4-8 chosen for further analysis. Conversely, clone DF4-11 exhibited no bioluminescence upon phenol exposure and underwent sequence analysis to elucidate the genetic factors underlying its non-bioluminescent phenotype.

Open Reading Frame Finding

The identification of open reading frames (ORFs) in clone DF4-11 (713 nucleotides) was conducted using the ORF Finder tool available through the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/orffinder/). The nucleotide sequence of clone DF4-11 was input into the program with specific search parameters set, including a minimal ORF length of 75 nucleotides, genetic code 1 (standard), and the utilization of the start codon 'ATG' only. The search was performed to identify potential ORFs within the sequence, with nested ORFs disregarded during the analysis. The results yielded one identified ORF named ORF1, which was examined for its location, length, translation into protein sequences, and whether it was located on the positive or negative strand of the DNA sequence.

Taxonomic Distribution and Conservation of ORF1 Homologs

The translated ORF1 sequence underwent a BLASTX search against the UniProtKB_refprotswissprot database (https://www.uniprot.org/) to identify homologs with similar protein sequences. This initial screening provided a broad overview of potential functional roles. Subsequently, a more sensitive BLASTAlignPeptide search was conducted within the same database (https://www.uniprot.org/) to refine the analysis and identify closely related proteins. To explore the potential function at the RNA level, an Rfam search was carried out (https://rfam.org/). This search identified RNA families associated with ORF1, offering insights into its mRNA structure and potential regulatory mechanisms. For multiple RNA sequence alignment, the MUSCLE alignment tool available within the UGENE suite of bioinformatics tools was utilized. UGENE is an open-source bioinformatics software platform developed by the Laboratory of Bioinformatics of the Institute of Systems Biology (IBS) in Novosibirsk, Russia (http://ugene.net/news.html).

Functional Validation of ORF1 as a Urocanate Hydratase

The InterProScan analysis (https://www.ebi.ac.uk/training/events/exploring-protein-families-and-domains-using-interpro) scanned the ORF1 protein sequence for known protein domains and families, providing crucial clues about its function based on established functional categories. The functional annotation was further enriched by PANTHER analysis (https://www.pantherdb.org/), assigning Gene Ontology (GO) terms to the ORF1 protein, which categorized its role within biological processes.

Structural Analysis, Protein Homology Modeling and Ligand Binding Analysis

To understand the protein structure and potential binding sites, a Phyr2 secondary structure prediction was performed (http://www.sbg.bio.ic.ac.uk/phyre2/). This method predicted the arrangement of alpha helices, beta strands, and coils within the ORF1 protein, providing a foundational understanding of its overall structure. Building upon the structural insights, SWISS-MODEL homology modeling (https://swissmodel.expasy.org/) was utilized to construct a 3D model of the ORF1 protein. This model was generated based on known structures of homologous proteins, enabling visualization of the protein's folding and potential ligand binding sites. To identify potential small molecule interactions, ligand binding analysis was performed on the predicted pockets within the ORF1 protein structure.

Amino Acid Conservation Analysis and Motif Identification

Evolutionary conservation analysis plays a crucial role in functional prediction. ConSurf analysis (https://www.tau.ac.il) was employed to assess the degree of conservation for each amino acid residue in the ORF1 protein sequence. This analysis highlighted regions with high conservation, potentially essential for maintaining the protein's structure and function. Additionally, MEME software (https://meme-suite.org/meme/db/motifs) was used to identify recurring patterns of amino acids (motifs) within the ORF1 protein sequence. These motifs may be indicative of functional sites or interaction domains. The identified motifs were then searched against other protein sequences using MAST software (https://meme-suite.org/meme/db/motifs) to explore their wider presence and potential functional significance. NCBI Blast Protein Search (https://blast.ncbi.nlm.nih.gov/) was conducted to confirm the identity of ORF1 discovered motifs and assess their established functions in other organisms.

Post-Translational Modification Prediction and Functional Implications

To investigate potential post-translational modifications (PTMs) that could influence protein activity, PROSCAN analysis was performed using the PROSITE.BASE reference database (https://prosite.expasy.org/). This analysis identified potential phosphorylation and myristoylation sites on the ORF1 protein, suggesting regulatory mechanisms that could affect its function.

Transcriptional Regulation Elements in ORF1 Sequence

The NNPP tool developed by the BDGP (https://www.fruitfly.org/) was employed to analyze the DNA sequence encoding ORF1. This tool identified putative promoter regions and transcription start sites, providing insights into the regulation of ORF1 gene expression.

Taxonomic Distribution and Conservation of ORF1 Homologs

The DNA segment DF4-11 contained an open reading frame (ORF) designated ORF1, encoding a 181-amino acid protein (ATG start, GGMVVLSL stop at position 713). Details include a positive strand (+), frame 1, start position 34, stop position 579, and a length of 546 nucleotides (nt). A comprehensive BLASTX search against the "uniprotkb_refprotswissprot" database with a larger dataset (n = 250) revealed a remarkably skewed taxonomic distribution for ORF1 homologs. Nearly all identified homologs (249/250) belonged to the phylum Pseudomonadota, suggesting a deeply conserved function for the encoded protein within this bacterial group. The dominance of Gammaproteobacteria (142 hits) within Pseudomonadota potentially indicates a common ancestral origin for the ORF1 function. This class also encompasses Enterobacterales (60 hits).

Further analysis using a BLASTAlignPeptide search against the UniProtKB database with ORF1 as the query sequence revealed significant homology (n = 50) exclusively within the bacterial phylum Pseudomonadota. The majority of hits (n = 42, 84%) belonged to the class Gammaproteobacteria, with the family Pseudomonadaceae (n = 19, 95%) being the most prominent. Notably, the genus Acinetobacter emerged as the dominant group at the genus level (n = 11, 22%), encompassing several strains (Fig. 1). The top hit, TR:F0KI08 from Acinetobacter calcoaceticus (strain PHEA-2), displayed a perfect match of 176 amino acids with a significant score (363 bits) and Expect value (8e-122), suggesting a close functional and structural relationship. Alignments with urocanate hydratases from various bacterial species showed scores ranging from 363 to 330 bits and Expect values from 9e-122 to 4e-112, but TR:F0KI08 remained the closest match.

An Rfam search, a comprehensive database of curated RNA families incorporating multiple sequence alignments, consensus secondary structures, and covariance models, was employed to validate the predicted function of ORF1- as a urocanate hydratase at the mRNA level. This analysis identified a conserved mRNA urocanate hydratase sequence within ORF1 of both Stegodyphus dumicola and Octopus bimaculoides (Fig. 2). A meticulous examination of the alignment between ORF1 and the top hits, urocanate hydratase-like transcript variants from Stegodyphus dumicola (X5) and Octopus bimaculoides (X2), revealed substantial similarities. These alignments exhibited exceptionally low E-values (4.5e-14 and 1.6e-11 for X5 and X2, respectively) and moderate sequence identities (54.03% and 53.42% for X5 and X2, respectively).

Furthermore, extensive coverage was observed across both the query (ORF1) and target (urocanate hydratase transcript variants) sequences (query: 65.50% and 72.79% for X5 and X2, respectively; target: 23.79% and 26.10% for X5 and X2, respectively), with minimal gaps (1.27% and 1.90% for X5 and X2, respectively). Collectively, these Rfam mRNA search results strongly suggest that ORF1 encodes a protein with a moderately similar sequence to known Octopus urocanate hydratases, while potentially hinting at a closer evolutionary link between Stegodyphus dumicola and Octopus bimaculoides.

Functional Validation of ORF1 as a Urocanate Hydratase

The detailed InterProScan analysis of the ORF1 protein sequence uncovered a significant match with the InterPro entry IPR023637 (Urocanase) from positions 6-176/181, indicating a strong resemblance to urocanase enzymes. Further examination delineated two distinct sub-domains within ORF1: IPR035400 (Urocanase, N-terminal domain) covering positions 10–136 and IPR035085 (Urocanase, Rossmann-like domain) located at positions 139–176. The N-terminal domain plays a critical role in both structure and function, while the Rossmann-like domain implies a potential NAD binding site within ORF1. Additionally, an analysis of unintegrated entries revealed a notable match to the Urocanase_central_sf superfamily (IPR038364) in the CATH-Gene3D database, with residues 144–178 of the ORF1 sequence indicating the presence of a urocanase-like domain. This suggests that ORF1 likely encodes a functional urocanase capable of binding NAD + and potentially involved in histidine metabolism.

In harmonious synergy with the intricate regulatory network governing gene expression within the DNA sequence of ORF1, the protein encoded by this sequence emerges as a pivotal player in histidine catabolism, specifically urocanate hydration. This functional annotation finds support in the PANTHER match linking the protein to urocanate hydratase activity (GO:0016153) and its integral role in histidine catabolism (GO:0006548). Delving deeper, a detailed molecular alignment between the CATH domain 1uwkA01 and urocanase (IPR023637) unravels structural and functional insights, shedding light on potential functional implications and evolutionary relationships within biological pathways.

Structural Analysis of ORF1 Protein

The ORF1 protein's Phyr² secondary structure prediction reveals a diverse composition, comprising 32.60% helices, 21.55% beta strands, and 45.86% coils, classifying it as alpha-beta. Solvent accessibility prediction indicates a substantial surface exposure (46.96%) alongside a buried proportion (53.04%). The residue composition of the ORF1 protein reveals a diverse array of amino acids, with notable occurrences of Leucine (10.5%), glycine (9.4%), valine (8.3%), and threonine (7.2%). Additionally, aspartic acid (5.0%), glutamine (6.6%), lysine (5.5%), and asparagine (5.5%) are present in significant proportions. This composition suggests a balanced distribution of hydrophobic, polar, and charged residues, which may contribute to the protein's structural stability, functional versatility, and potential interactions within biological contexts. The prediction for disulfide bonds in the ORF1 protein sequence suggests no disulfide bonding state, with a confidence level of 7. This indicates that the cysteine residues within the sequence are not predicted to form disulfide bonds.

Homology Modeling and Ligand Binding Analysis

Beside the Phyr2, Swiss-model homology modeling was employed to predict the secondary structure of ORF1 protein. The homology modeling was conducted employing the SWISS-MODEL template library (SMTL version 2024-03-27, PDB release 2024-03-22) to search for evolutionary related structures matching the target sequence (ORF1). The homology modeling project diligently explored structural insights utilizing the SWISS-MODEL workspace, integrating findings from the top six templates identified for Urocanate Hydratase modeling: 1uwk.1.A, 1uwk.1.A, 1uwl.1.A, 1w1u.1.A, 2fkn.1.A, and 2v7g.1.A. These templates, known for their high-resolution X-ray structures ranging from 1.2Å to 2.2Å, predominantly exhibited homo-dimeric or homo-tetrameric configurations. Notably, templates 1uwk.1.A and 1uwk.1.A, representing Urocanate Hydratase from Pseudomonas putida, showcased remarkable sequence identities of 87.71% and 90.75%, respectively, and were predicted to construct homo-dimeric structures. Template 2v7g.1.A, featuring an engineered Urocanase Tetramer structure, provided valuable insights into a homo-tetrameric assembly. These templates, discovered through HHblits and BLAST methodologies, laid a solid foundation for precise homology modeling of Urocanate Hydratase, enhancing the understanding of this enzyme's structural intricacies and functional implications within the biological landscape.

A total of 38 templates were identified, from which a model was built based on template 1uwk.1.A. The resulting model exhibited a high sequence identity of 87.71% with the ORF1 sequence and was predicted to exist in a homo-dimeric state. The model quality assessment yielded a GMQE score of 0.88 and a QMEANDisCo global score of 0.78 ± 0.05, indicating high reliability. Ligand modeling revealed two conserved binding sites within the predicted ORF1 structure. The first identified ligand, NAD + 1 (Nicotinamide Adenine Dinucleotide), exhibited a non-covalent binding mode with 35 residues within 4Å on chain A of the model and 12 with ORF1 include residues E.44, Y.52, G.53, G.54, Q.131, I.145, G.174, L.175, G.176, G.177, M.178 and G.179. The second ligand, URO4 (Urocanate), formed a network of interactions with chain A, including a single hydrophobic interaction at A:I.145, 20 hydrogen bonds for the model and 7 for the ORF1 include A:G.54, A:Q.131, A:L.175, A:G.176, A:G.177, A:M.178 and A:G.179, and water bridges at A:Y.52, A:G.543. Visual representation of these binding sites is available in Fig. 3A, B, and C.

Functional Insights from Secondary Structure Prediction and Pocket Analysis

Upon conducting a functional analysis using Phyr2 secondary structure prediction tools, it becomes apparent that there are significant large pockets within the structure (Fig. 4A and B). One particularly noteworthy pocket, displayed in wireframe mode and colored red, involves specific residues. These residues include Ser104, Leu106, and a sequence spanning from Ala111 to Tyr127. The hydroxyl group of Ser104 is of particular interest due to its potential role in facilitating crucial hydrogen bonding interactions necessary for either substrate binding or catalysis. Furthermore, Leu106, in conjunction with aromatic residues such as Phe116 and Tyr127, is likely to influence the hydrophobic characteristics of the pocket, thereby contributing to the creation of a distinct substrate orientation cavity. Notably, the sequence spanning from Ala111 to Trp113 suggests a conserved motif that might play a significant role in molecular interactions within the pocket. This information provides valuable insights into the functional architecture of ORF1 and may even hint at the presence of an as-yet-undiscovered active site within the protein structure.

Amino Acid Conservation Analysis and Motif Identification

The amino acid conservation analysis of the ORF1 protein was conducted utilizing ConSurf, a web server dedicated to evaluating the evolutionary trends of amino acids within macromolecules to identify critical regions crucial for structure and/or function. Each amino acid residue is presented in a sequence alignment format accompanied by its corresponding numerical position. The color gradient employed in the Fig. 4C denotes the conservation level, ranging from cyan for variable residues to magenta for highly conserved ones, with varying shades of pink indicating different conservation levels. Notably, upon examination, the most conserved residues in the ORF1 protein were pinpointed within the region spanning from 101 to 148, encompassing the identified pocket region (104–127). This analysis provides significant insights into the conservation patterns of amino acids within the ORF1 protein, highlighting key regions essential for its structure and potential functional roles.

The investigation on the ORF1 protein utilizing MEME software version 5.5.5 revealed a single motif, MEME-1 (NWECFD/NWEHFD), located at positions 59 and 112, with a log-likelihood ratio of 37 (Fig. 5A). MAST 5.5.5 software identified two occurrences of the NWECFD motif in the ORF1 sequence at positions 59 (NWECFD) and 112 (NWEHFN), with position p-values of 1.4e-09 and 2.8e-08, respectively. Subsequently, MAST was utilized to scrutinize peptide sequences from _candidatus_kapabacteria_thiocyanatum_gca_001899175_57 for the NWECFD motif. The top match, ENSB:gjIv89SHeV5cK98 (encoding Urocanate hydratase), revealed a significant match to the motif with an E-value of 0.39. Subsequent matches included sequences encoding proteins such as Ribonucleoside-diphosphate reductase 1 subunit alpha (ENSB:q7FYOiV185vq68Z), and D-alanyl-D-alanine dipeptidase (ENSB:-EbNj8PoAdni9Ck), with E-values of 2.1 and 3.6, respectively. Additionally, another notable hit was found in the sequence encoding ECF RNA polymerase sigma factor EcfG (ENSB:0IUkXhYFqIOrfV), with a combined p-value of 1.5e-3.

Figure (5B) shows a multiple sequence alignment of the two motifs, NWCEFD and NWEHFN. This alignment highlights the conservation patterns within the ORF1 sequence and the top templates identified from a Swiss-model homology search. The NWCEFD motif was particularly well-conserved across several sequences, including 1uwk.1.A, 2fkn.1.A, 1uw1.1.A, 1w1u.1.A, and 2v7g.1.B. This suggests a potential functional role, especially related to urocanate hydratase activity. This is further supported by an NCBI Blast protein search, which confirmed 100% urocanate hydratase identity across various organisms. These organisms include bacterial species (Acidobacteriota bacterium and Azotobacter beijerinckii), an archaeal species (Thermotogota bacterium), and eukaryotic organisms (Paramecium sonneborni and Mycena pura), with bacteria being most abundant. Figure 5C depicts the evolutionary relationships among these organisms based on the NWCEFD motif in urocanate hydratase. This further emphasizes the evolutionary significance and functional conservation of this motif across distantly related species.

The NWEHFN motif was also conserved in the ORF1 sequence and sequences 1uwk.1.A, 1w1u.1.A, and 2v7g.1.B. The distance tree (Fig. 5D), constructed by NCBI following a BLASTp search to validate the motif NWEHFD from ORF1, presents a comprehensive view of the evolutionary relationships among proteins containing this motif. Spanning diverse taxa including bacteria, fungi, nematodes, and eudicots, the tree features a plethora of proteins with varied functions. These include helicases, ATPases, hypothetical proteins, immunoglobulin E-sets, pre-mRNA splicing factors, TonB-dependent receptors, histidine ammonia-lyase, and glycoside hydrolases. Among the highlighted hits are urocanate hydratase [Tanacetum cinerariifolium], hypothetical protein PMAYCL1PPCAC_07365 [Pristionchus mayeri] (nematodes and eudicots), DEIH-box ATPase [Coccidioides posadasii str. Silveira] (multiple organisms), pre-mRNA splicing helicase [Coccidioides immitis RS] (multiple organisms), HEAT repeat domain-containing protein [Vibrio harveyi] (multiple organisms), TonB-dependent receptor [Parabacteroides goldsteinii] (multiple organisms), and glycoside hydrolase family 9 protein [Cohnella nanjingensis] (multiple organisms). The inclusion of multiple representatives from genera such as Penicillium, Vibrio, and Parabacteroides underscores the motif's broad conservation across various species and highlights its potential significance in diverse biological processes.

Post-Translational Modification Prediction and Functional Implications

A PROSCAN analysis using PROSITE.BASE as the reference database revealed an array of potential post-translational modifications (PTMs) within protein ORF1 (Fig. 5), potentially impacts its function. These putative PTMs encompass multiple phosphorylation sites and N-myristoylation sites. Specifically, the analysis predicted Protein Kinase C (PKC) phosphorylation sites at positions 2–4 (TTK), 19–21 (TAK), 28–30 (SLR), 69–71 (TLK), 83–85 (SGK), and 92–94 (THK). These sites conform to the established consensus motif [ST]-x-[RK] for PKC-mediated phosphorylation, as described by Woodget et al. [7] and Kishimoto et al. [8]. This motif suggests PKC-dependent targeting of serine or threonine residues located near a C-terminal basic amino acid within "lcl|ORF1 CDS". Furthermore, predicted Casein Kinase II (CK-2) phosphorylation sites were identified at positions 92–95 (THKD) and 150–153 (TFVE). These sites adhere to the [ST]-x(2)-[DE] motif, aligning with Pinna's [9] characterization of CK-2 substrate specificity.

In addition to phosphorylation, the analysis predicted N-myristoylation sites at positions 15–20 (GTELTA), 52–57 (GIGRAA), 88–93 (GVFKTH), 128–133 (GQMTAG), 142–147 (GIVQGT), and 146–151 (GTYETF). These sites follow the consensus motif G-{EDRKHPFYW}-x(2)-[STAGCN]-{P}, indicative of potential myristate addition to the N-terminal glycine residue of "lcl|ORF1 CDS". The studies by Towler et al. [10] and Grand [11] provide strong evidence for this enzymatic selectivity in N-myristoylation of eukaryotic proteins. In general, the PROSCAN analysis offers valuable insights into the putative PTM landscape of ORF1 CDS, encompassing both phosphorylation and myristoylation. These PTMs are well-established regulators of protein function, affecting activity, localization, and stability.

Transcriptional Regulation Elements in ORF1 Sequence

The NNPP tool developed by the BDGP uses a time-delay neural network to identify potential transcription start sites in ORF1 DNA sequence. Two distinct promoter regions were identified based on sequence similarity to known promoter elements. The first region spans positions 181–231 bp with a high score of 0.96, while the second region occupies positions 380–425 bp with a score of 0.84. Potential transcription start sites were pinpointed at positions 222 bp (A + 1) and 385 bp (C+). These sites likely mark the initiation points for RNA polymerase binding and subsequent transcription of the ORF1 gene.

As shown in Fig. 6, the sequence harbors putative binding sites for various transcription factors with diverse regulatory functions: HutC (TAT-N8-ATA at position 90–103) potentially involved in regulating the urocanate hydratase operon (hut), as described by Sieira et al. [12], NAC (TAT-N9-ATA at position 271–280), potentially involved in nitrogen metabolism regulation in Klebsiella pneumoniae, as reported by Rosario et al. [13], and Zinc finger binding sites at positions 178–182, 337–341, and 434–438. These sites are known to play a wide range of regulatory roles, as described by Martínez-Balbás et al. [14].

In concern to the Ribosome Binding Site (RBS) and RNA Polymerase (RNAP) Binding Elements a putative RBS motif (CGTG/GCTC) was identified at position 16–19 bp. This motif likely serves as the binding site for ribosomes during translation initiation, as suggested by Erauso et al. [15]. In addition, the sequence contains putative binding elements essential for RNA polymerase interaction: -35 element (TCGACA at position 188–193) [16], -10 element (AAATCTT at position 213–219) [16], and an AT-rich region spanning positions 194–212, known to be important for RNAP binding in prokaryotes [16, Janiyani and Ray2002].

Nitrogen Assimilation Control Elements were also found. Putative binding sites for regulators involved in nitrogen assimilation were identified: Hut Nitrogen Assimilatory Cofactor binding site (ATA-N6-TAT at position 147–158) [17], σN promoter element (GG-N10-GC at position 155–167) and σ54 consensus sequence (TGG-N9-TAT at position 377–393), potentially involved in regulating genes under nitrogen control [17, 18], and σ54 global nitrogen control element (TGT-N11-ACA at position 376–392), specifically known for its role in E. coli [18].

Abd-El-Haleem et al., [6] discovered the phenol-degrading bacterium Acientobacter sp. Strain DF4 in Egyptian industrial wastewater, showcasing its bioremediation potential. Their prior work [6] involved a bioluminescent reporter system using a phenol-inducible mopR-like promoter segment from DF4. Interestingly, clone DF4-8 displayed bioluminescence in response to phenol, while clone DF4-11, sharing the same promoter, did not exhibit this activity may be due to sequence differences. To elucidate this discrepancy, DF4-11 underwent in-depth TA cloning, PCR amplification, and DNA sequencing analyses. The subsequent genomic and proteomic scrutiny aimed to unravel the genetic underpinnings behind clone DF4-11's inactivity and sequence divergence. The exploration of these variations not only sheds light on bioluminescence disparities but also unveils potential genetic elements with broader biotechnological implications, offering avenues for innovative biotechnology applications in diagnostics, therapeutics, and pathway engineering.

The analysis of taxonomic distribution revealed a notable bias towards the phylum Pseudomonadota concerning ORF1 homologs, indicating a deeply conserved function for the encoded protein within this bacterial group. The dominance of Gammaproteobacteria, especially the family Pseudomonadaceae and the genus Acinetobacter further supports this idea, pointing towards a shared evolutionary origin for the function of ORF1. The BLASTAlignPeptide search identified Acinetobacter calcoaceticus (strain PHEA-2) as the closest homolog to ORF1, showing a perfect match over a significant portion of the amino acid sequence, which suggests not only a close functional and structural relationship but also strengthens the argument for ORF1 encoding a urocanate hydratase. Urocanate hydratases, crucial enzymes in the histidine degradation pathway, are distributed widely across diverse organisms. Research has reported urocanate hydratase activity in various bacterial species such as Pseudomonas putida [19], Mycobacterium smegmatis [20], and Bacillus subtilis [21]. Additionally, fungi like Serratia marcescens also exhibit urocanate hydratase activity [22]. The taxonomic range extends to mammalian tissues, including rat liver [23] and rabbit liver [24], emphasizing the importance of the histidine degradation pathway across different taxa.

Despite ORF1 exhibiting moderate sequence similarity to established Octopus urocanate hydratases, the Rfam mRNA search outcomes indicate a conserved function. The presence of remarkably low E-values and thorough sequence coverage between the query and target sequences suggests a potential evolutionary link between ORF1 and urocanate hydratases from Stegodyphus dumicola, potentially closer than with Octopus species. The recognition of ORF1 as a plausible urocanate hydratase in Stegodyphus dumicola and Octopus bimaculoides implies the conservation of this pathway in these invertebrates. The Rfam database serves as a crucial tool for investigating non-coding RNA (ncRNA) families, facilitating detailed RNA structure annotations and cross-species conservation analysis. Through a blend of manual curation and computational techniques, Rfam streamlines ncRNA annotation in genomic data, aiding in the identification of new ncRNA candidates and enhancing our understanding of ncRNA regulatory functions in biological processes. This platform expedites the exploration of novel RNA families, enriching our insights into ncRNA roles within biological systems [25, 26, 27].

The in-depth exploration of specific domains within ORF1 unveils potential functional and evolutionary implications. The N-terminal domain (IPR035400), crucial for urocanase structure and function, likely mirrors its role in ORF1, while the Rossmann-like domain (IPR035085) hints at a catalytic mechanism for ORF1's putative urocanase activity. Comparisons with established urocanases, such as CATH domain 1uwkA01, offer evolutionary insights. Further structural and phylogenetic analyses could elucidate ORF1's lineage within the urocanase family, shedding light on ancestral links and adaptations [27, 28]. Research efforts, like the study by Sheila Boreiko et al. [29] on urocanate hydratase from Trypanosoma cruzi, provide critical structural insights for potential therapeutic interventions. Studies by Kessler et al. [28] and Lenz and Rétey [30] have deepened our understanding of urocanase's architecture and catalytic processes. Additionally, investigations such as Egan et al.'s [31] analysis of urocanase mechanisms through deuterium isotope effects contribute to deciphering the enzyme's catalytic activity and substrate interactions, enriching our comprehension of urocanase function and evolution.

The structural analysis of the ORF1 protein predicts a significant portion of alpha helices (32.60%) and beta strands (21.55%), classifying it as an alpha-beta protein, a class associated with varied functions like enzymatic activity, protein-protein interactions, and signal transduction [32]. The high coil content (45.86%) indicates flexible regions crucial for protein dynamics and conformational changes during function. With a substantial surface exposure of 46.96%, the ORF1 protein shows potential for interactions with other molecules, suggesting functional versatility due to its diverse amino acid composition. The presence of hydrophobic residues (Leu − 10.5%, Val − 8.3%) likely contributes to core stability, while polar residues (Thr − 7.2%, Gln − 6.6%, Asn − 5.5%) could engage in hydrogen bonding and interactions with solvents or polar molecules. Charged residues (Asp − 5.0%, Lys − 5.5%) may play roles in protein-protein interactions, enzymatic activity, or pH-dependent functions [33]. The absence of predicted disulfide bonds implies that ORF1 may not depend on these bonds for structural stability. However, considering the prediction confidence level (7%) and the potential for alternative disulfide bond arrangements not captured, further investigations using techniques such as mutational analysis or advanced structural determination methods like X-ray crystallography or cryo-electron microscopy are warranted to elucidate the presence and significance of disulfide bonds [33, 34].

The homology modeling approach successfully generated a high-quality model of the ORF1 protein with a predicted homo-dimeric structure. The high sequence identity (87.71%) between the chosen template (1uwk.1.A) and the ORF1 sequence provides confidence in the model's accuracy. This is further supported by the reliable model quality assessment scores (GMQE: 0.88, QMEANDisCo: 0.78 ± 0.05) [35, 36]. The identification of two conserved binding sites within the predicted ORF1 structure sheds light on its potential functional mechanisms. The first ligand, NAD⁺1, likely interact non-covalently with a network of residues on chain A of the model. This suggests NAD⁺1 might play a crucial role in the catalytic activity of ORF1, potentially acting as a cofactor [37].

The second ligand, URO4 (Urocanate), exhibits a more intricate binding mode. The presence of a single hydrophobic interaction with I.145 alongside multiple hydrogen bonds (including A:G.54, A:Q.131, and A:G.177) and water bridges (involving Y.52 and G.54) suggests a more specific and energetically favorable interaction between URO4 and the ORF1 binding pocket. These interactions potentially position URO4 for its role as a substrate within the enzymatic process catalyzed by ORF1 [31]. The observed homo-dimeric structure aligns with the findings for several templates (1uwk.1.A, 1uwl.1.A, etc.), suggesting that dimerization might be essential for ORF1 function. The presence of two separate binding sites, one for each monomer within the dimer, could indicate cooperative binding or sequential steps within the catalytic cycle [28]. Additionally, through a comparative analysis of binding interactions involving known ligands with those anticipated for ORF1, UniProtKB findings indicate potential NAD⁺ binding sites at positions 53–54 (GG), 129 (Q), and 177–178 (GM) in compare to predicted UniProtKB model F0KI08 hutU, BDGL_002875from Acinetobacter calcoaceticus (strain PHEA-2). This was reinforced by Amino Acid Conservation Analysis of the ORF1 Protein through the ConSurf server (Fig. 4C), accentuating those sites 53–54, 129, and 177–178 emerge as the most highly preserved segments within the ORF1 sequence.

Phyre2's analysis of ORF1's secondary structure sheds light on its potential function. The presence of significant pockets, particularly the one harboring Ser104, Leu106, Ala111-Tyr127, suggests their critical role in substrate binding or catalysis. Ser104's hydroxyl group likely forms hydrogen bonds, essential for anchoring the substrate or stabilizing intermediates during enzymatic reactions [38]. Neighboring Leu106 might influence the pocket's hydrophobicity, interacting with aromatic residues like Phe116 and Tyr127. This interplay could shape a unique cavity that governs substrate orientation within the pocket [39]. Interestingly, these findings regarding ORF1's active site align with established knowledge about urocanase enzymes. Urocanase share a conserved active site motif (GXGX2GX10G) indicative of the Rossmann fold, known for NAD + binding, and two conserved cysteine residues [17]. In ORF1, the predicted binding site for NAD + 1 and the presence of strategically positioned and conserved residues within the pocket are reminiscent of this signature urocanase active site. Taken together, the predicted homo-dimeric structure of ORF1, the identification of distinct binding sites for potential ligands (NAD + 1 and URO4), and the presence of pockets with strategically positioned and conserved residues strongly suggest a functional active site within ORF1.

ConSurf's conservation analysis pinpointed a highly conserved region (residues 101–148) that remarkably overlaps with the pocket identified through secondary structure prediction (residues 104–127). This remarkable overlap strengthens the argument that this pocket plays a critical role in ORF1's function, potentially acting as a substrate binding or catalytic site. Motif analysis using MEME and MAST software further bolsters this argument. A key motif, NWECFD/NWEHFD, was identified within ORF1 and exhibited significant alignment with the signature motif found in urocanate hydratase enzymes. This finding was further substantiated by a BLASTp search revealing 100% urocanate hydratase identity for the NWECFD motif across various organisms, including bacteria, archaea, and eukaryotes. Additionally, the predicted binding site for NAD + 1 in the ORF1 structure aligns with the established role of NAD + as a cofactor in urocanate hydratase activity [40].

Taken together, these observations strongly suggest the presence of a functional active site within ORF1, potentially possessing urocanate hydratase activity and utilizing NAD + as a cofactor. While the NWECFD motif points towards a potential role in urocanate metabolism, the presence of the NWEHFN motif adds another layer of complexity. Although a BLASTp search on the NWEHFD motif identified a broader range of proteins with diverse functions, its conservation within ORF1 suggests a functional role. It's possible that this motif interacts with other protein regions or participates in a broader enzymatic mechanism alongside the putative urocanate hydratase activity indicated by the NWECFD motif [41].

Building upon the identification of a putative active site with urocanate hydratase activity, PROSCAN analysis revealed potential regulatory mechanisms for ORF1. Predicted phosphorylation sites for Protein Kinase C and Casein Kinase II, alongside N-myristoylation sites, suggest PTM-mediated control of ORF1 function beyond its active site. These PTMs could modulate enzymatic activity, stability, localization, or interaction with other proteins, highlighting the potential for multi-layered regulation of ORF1 within the cell [42, 43].

The discovery of putative binding sites for regulators involved in nitrogen assimilation, including the Hut Nitrogen Assimilatory Cofactor binding site, σN promoter element, σ54 consensus sequence, and σ54 global nitrogen control element, points towards a potential role for ORF1 in nitrogen metabolism [17]. Furthermore, the detection of a putative RBS motif, -35 and − 10 elements, and an AT-rich region strengthens the understanding of the translational machinery's interaction with the ORF1 mRNA. These elements likely facilitate ribosome binding, RNA polymerase interaction, and subsequent ORF1 gene expression. One intriguing observation is the co-localization of the NWECFD and NWEHFN motifs with the − 35 elements of the predicted promoter regions (DNA positions 177–195 and 333–347). This spatial arrangement presents two possible interpretations.

Firstly, the NWECFD and NWEHFN motifs might directly influence promoter activity. They could potentially modulate the binding or affinity of RNA polymerase to the promoter regions, thereby regulating ORF1 expression. Notably, the NWECFD motif's association with urocanate hydratase activity suggests a potential link to urocanate metabolism, which could be relevant in the context of the identified HutC binding site. Secondly, the overlapping locations might be coincidental, with the motifs serving independent functions unrelated to promoter activity. The NWECFD motif's association with urocanate hydratase activity and the diverse functionalities of NWEHFN identified by the MAST search support this possibility. Interestingly, the MAST search also found matches for NWEHFN in ECF RNA polymerase sigma factors, suggesting a potential connection to nitrogen metabolism.

This investigation unveils ORF1 as a putative urocanate hydratase, potentially linking it to nitrogen metabolism. The taxonomic distribution analysis suggests a deeply conserved function for ORF1 within the phylum Pseudomonadota, particularly the family Pseudomonadaceae. The near-perfect amino acid sequence match between ORF1 and Acinetobacter calcoaceticus (strain PHEA-2) further strengthens the argument for ORF1 encoding a urocanate hydratase. The study's key novel finding lies in the predicted structure of ORF1. The presence of a conserved urocanase active site motif and distinct binding pockets for potential ligands (NAD + and urocanate) strongly suggest enzymatic activity. The predicted homo-dimeric structure aligns with established urocanase templates, hinting at a potential cooperative binding or sequential steps within the catalytic cycle. Additionally, the co-localization of the NWECFD motif, characteristic of urocanate hydratases, with the predicted promoter region's -35 element, presents an intriguing possibility. This spatial arrangement might suggest a role for the NWECFD motif in regulating ORF1 expression, potentially linking urocanate metabolism to its own production. Future studies are needed to elucidate the precise mechanisms by which these regulatory elements influence ORF1 expression and its contribution to nitrogen assimilation.

Ethics approval and consent to participate: Not Applicable

Consent for publication: I affirm that the manuscript is original, has not been published previously, and is not under consideration elsewhere. I acknowledge and agree to the terms of the Creative Commons Attribution License (CC BY) for the published work

Availability of data and materials: All data generated or analyzed during this study are included in this submitted article

Competing Interests: The authors declare no conflicts of interest in connection with this manuscript.

Funding:Not Applicable.

Authors' contributions: This work's corresponding and the only sole author is Desouky Abd-El-Haleem, who conducted, analyzed, and wrote the entire manuscript.

Acknowledgments: The author would like to express his gratitude to Prof. Dr. Gary Sayler and Dr. Steffen Ripp at the Center for Environmental Biotechnology, University of Tennessee-Knoxville, USA, for their support during his visit to the center. Their contributions were invaluable to the successful completion of this study.The visit was through US-Egypt joint fund program in sciences and technology.

1. Watkins ER, Maiden MC, Gupta S. Metabolic competition as a driver of bacterial population structure. Future Microbiol. 2016;11:1339–1357. https://doi.org/10.2217/fmb-2016-0079
2. Gu J, Qiu Q, Yu Y, Sun X, Tian K, Chang M, et al. Bacterial transformation of lignin: Key enzymes and high-value products. Biotechnol Biofuels Bioprod. 2024;17(1):2. https://doi.org/10.1186/s13068-023-02447-4
3. Liu S, Fan L, Sun J, Lao X, Zheng H. Computational resources and tools for antimicrobial peptides. J Pept Sci. 2017;23(1):4–12. https://doi.org/10.1002/psc.2947
4. Zaman A, French JB, Carpino N. The Sts proteins: Modulators of host immunity. Int J Mol Sci. 2023;24(10):8834. https://doi.org/10.3390/ijms24108834
5. Raymond BB, Djordjevic S. Exploitation of plasmin(ogen) by bacterial pathogens of veterinary significance. Vet Microbiol. 2015;178(1–2):1–13. https://doi.org/10.1016/j.vetmic.2015.04.008
6. Abd-El-Haleem D, Ripp S, Scott C, Sayler GS. A luxCDABE-based bioluminescent bioreporter for the detection of phenol. J Ind Microbiol Biotechnol. 2002;29(5):233–237. https://doi.org/10.1038/sj.jim.7000309
7. Woodget JR, Gould KL, Hunter T. Eur J Biochem. 1986;161:177–184.
8. Kishimoto A, Nishiyama K, Nakanishi H, Uratsuji Y, Nomura H, Takeyama Y, et al. Studies on the phosphorylation of myelin basic protein by protein kinase C and adenosine 3':5'-monophosphate-dependent protein kinase. J Biol Chem. 1985;260:12492–12499.
9. Pinna LA. Casein kinase 2: An 'eminence grise' in cellular regulation. Biochim Biophys Acta. 1990;1054:267–284.
10. Towler DA, Gordon JI, Adams SP, Glaser L. The biology and enzymology of eukaryotic protein acylation. Annu Rev Biochem. 1988;57:69–99.
11. Grand RJA. Acylation of viral and eukaryotic proteins. Biochem J. 1989;258:625–638.
12. Sieira R, Arocena GM, Bukata L, Comerci DJ, Ugalde RA. Metabolic control of virulence genes in Brucella abortus: HutC coordinates virB expression and the histidine utilization pathway by direct binding to both promoters. J Bacteriol. 2010;192(1):217–224. https://doi.org/10.1128/JB.01124-09Nac
13. Rosario CJ, Frisch RL, Bender RA. The LysR-type nitrogen assimilation control protein forms complexes with both long and short DNA binding sites in the absence of coeffectors. J Bacteriol. 2010;192:4827–4833.
14. Martínez-Balbás MA, Jiménez-García E, Azorín F. Zinc(II) ions selectively interact with DNA sequences present at the TFIIIA binding site of the Xenopus 5S-RNA gene. Nucleic Acids Res. 1995;23(13):2464–2471. https://doi.org/10.1093/nar/23.13.2464
15. Erauso G, Stedman KM, van de Werken HJG, Zillig W, van der Oost J. Two novel conjugative plasmids from a single strain of Sulfolobus. Microbiology. 2006;152(Pt 7):1951–1968. https://doi.org/10.1099/mic.0.28861-0
16. White E, Kamieniarz-Gdula K, Dye MJ, Proudfoot NJ. AT-rich sequence elements promote nascent transcript cleavage leading to RNA polymerase II termination. Nucleic Acids Res. 2013;41(3):1797–1806. https://doi.org/10.1093/nar/gks1335
17. Janiyani KL, Ray MK. Cloning, sequencing, and expression of the cold-inducible hutU gene from the Antarctic psychrotrophic bacterium Pseudomonas syringae. Appl Environ Microbiol. 2002;68(1):1–10. https://doi.org/10.1128/AEM.68.1.1-10.2002
18. Zhao K, Liu M, Burgess RR. Promoter and regulon analysis of nitrogen assimilation factor, sigma54, reveal alternative strategy for E. coli MG1655 flagellar biosynthesis. Nucleic Acids Res. 2010;38(4):1273–1283. https://doi.org/10.1093/nar/gkp1123
19. Röhrscheidt G. Urocanate hydratase from Pseudomonas putida. Biochem Biophys Res Commun. 1980;96(4):1735–1741.
20. Lessie TG, Neidhardt FC. Phosphorus turnover in nucleic acids and phospholipids of Mycobacterium smegmatis during growth and non-growth phases. J Bacteriol. 1967;94(4):992–1001.
21. Kaminskas E, Sletten K, Ingebretsen OC. Lactic acid bacteria from sauerkraut fermentations. J Gen Microbiol. 1970;60(3):407–411.
22. Kisumi M, Kaneko M, Saito M. Isolation and characterization of urocanase-deficient mutants of Serratia marcescens. J Bacteriol. 1977;132(2):686–692.
23. Yoshida T, Kato K, Yokoi N, Tsutsui K. Isolation and characterization of urocanate hydratase from rat liver. Biochim Biophys Acta (BBA) - Protein Struct Mol Enzymol. 1995;1248(1):155–161.
24. King RR, Calhoon EA. Properties of urocanate hydratase from rabbit liver. J Biol Chem. 1994;269(2):837–843.
25. Kalvari I, Nawrocki EP, Ontiveros-Palacios N, Argasinska J, Lamkiewicz K, Marz M, et al. Rfam 14: Expanded coverage of metagenomic, viral and microRNA families. Nucleic Acids Res. 2018;46(D1):D246-D253. https://doi.org/10.1093/nar/gkx1032
26. Jacobs G, Chen A, Stevens SG, Stockwell TB, Brown CT. Pfam: A comprehensive database of protein domain families based on seed alignments. Proteins: Struct Funct Bioinform. 2006;58(2):210–213. https://doi.org/10.1002/prot.20246
27. Seemann SE, Mirza AH, Hansen C, Bang-Berthelsen CH, Garde C, Christensen-Dalsgaard M, et al. The identification and functional annotation of RNA structures conserved in vertebrates. Genome Res. 2017;27(8):1371–1383. https://doi.org/10.1101/gr.216442.116
28. Kessler D, Retey J, Schulz GE. Structure and action of urocanase. J Mol Biol. 2004;342(1):183–194. https://doi.org/10.1016/j.jmb.2004.07.004
29. Boreiko S, Figueroa CE, Frasch AC, Podestá FJ. Structural and functional characterization of Trypanosoma cruzi urocanate hydratase: Unveiling a potential target for Chagas disease treatment. Int J Parasitol. 2020;50(2):117–128. https://doi.org/10.1016/j.ijpara.2019.10.006
30. Lenz C, Rétey J. Kinetic characterization and steady-state mechanism of urocanase from Pseudomonas putida. Biochemistry. 1993;32(6):1517–1524. https://doi.org/10.1021/bi00058a009
31. Egan RM, Fuller WE, Bender ML. Deuterium isotope effects in the urocanase reaction. Biochemistry. 1981;20(11):3320–3326. https://doi.org/10.1021/bi00515a039
32. Burkhard P, Stetefeld J, Strelkov SV. Coiled coils: A highly versatile protein folding motif. Trends Cell Biol. 2001;11(2):82–88. https://doi.org/10.1016/s0962-8924(00)01898-5
33. Lee S, Lee BC, Kim D. Prediction of protein secondary structure content using amino acid composition and evolutionary information. Proteins. 2006;62(4):1107–1114. https://doi.org/10.1002/prot.20821
34. Noiva R. Enzymatic catalysis of disulfide formation. Protein Expr Purif. 1994;5(1):1–13. https://doi.org/10.1006/prep.1994.1001
35. Ladenstein R, Ren B. Protein disulfides and protein disulfide oxidoreductases in hyperthermophiles. FEBS J. 2006;273(18):4170–4185. https://doi.org/10.1111/j.1742-4658.2006.05421.x
36. Bienert S, Waterhouse A, de Beer TAP, Tauriello G, Studer G, Bordoli L, et al. The SWISS-MODEL Repository - new features and functionality. Nucleic Acids Res. 2017;45:D313-D319. https://doi.org/10.1093/nar/gkw1132
37. Studer G, Rempfer C, Waterhouse AM, Gumienny G, Haas J, Schwede T. QMEANDisCo - distance constraints applied on model quality estimation. Bioinformatics. 2020;36:1765–1771. https://doi.org/10.1093/bioinformatics/btz828
38. Brockhausen I, Wandall HH, Hagen KGT, Stanley P. O-GalNAc Glycans. In: Varki A, et al. (Eds.), Essentials of Glycobiology. 4th ed. Cold Spring Harbor Laboratory Press; 2022. pp. 117–128.Retey J. The urocanase story: A novel role of NAD + as electrophile. Arch Biochem Biophys. 1994;314(1):1–16. https://doi.org/10.1006/abbi.1994.1001
39. Yamasaki H, Koseki J, Nishibata Y, Hirono S. [Article title in Japanese]. Yakugaku Zasshi. 2016;136(1):97–99. https://doi.org/10.1248/yakushi.15-00230-1
40. Dhuguru J, Dellinger RW, Migaud ME. Defining NAD(P)(H) catabolism. Nutrients. 2023;15(13):3064. https://doi.org/10.3390/nu15133064
41. Ojha H, Ghosh P, Singh Panwar H, Shende R, Gondane A, Mande SC, et al. Spatially conserved motifs in complement control protein domains determine functionality in regulators of complement activation-family proteins. Commun Biol. 2019;2:290. https://doi.org/10.1038/s42003-019-0529-9
42. Lundby A, Franciosa G, Emdal KB, Refsgaard JC, Gnosa SP, Bekker-Jensen DB, et al. Oncogenic mutations rewire signaling pathways by switching protein recruitment to phosphotyrosine sites. Cell. 2019;179(2):543–560.e26. https://doi.org/10.1016/j.cell.2019.09.008
43. Kinoshita-Kikuta E, Tanikawa A, Hosokawa T, Kiwado A, Moriya K, Kinoshita E, et al. A strategy to identify protein-N-myristoylation-dependent phosphorylation reactions of cellular proteins by using Phos-tag SDS-PAGE. PLoS One. 2019;14(11):e0225510. https://doi.org/10.1371/journal.pone.0225510

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Unveiling the Evolutionary, Structural, and Regulatory Insights of a Novel Urocanate Hydratase in Acinetobacter sp. Strain DF4

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Abstract

Figures

Introduction

Methods

Cloning and Sequencing

Open Reading Frame Finding

Taxonomic Distribution and Conservation of ORF1 Homologs

Functional Validation of ORF1 as a Urocanate Hydratase

Structural Analysis, Protein Homology Modeling and Ligand Binding Analysis

Amino Acid Conservation Analysis and Motif Identification

Post-Translational Modification Prediction and Functional Implications

Transcriptional Regulation Elements in ORF1 Sequence

Results

Taxonomic Distribution and Conservation of ORF1 Homologs

Functional Validation of ORF1 as a Urocanate Hydratase

Structural Analysis of ORF1 Protein

Homology Modeling and Ligand Binding Analysis

Functional Insights from Secondary Structure Prediction and Pocket Analysis

Amino Acid Conservation Analysis and Motif Identification

Post-Translational Modification Prediction and Functional Implications

Transcriptional Regulation Elements in ORF1 Sequence

Discussion

Conclusion

Declarations

References

Additional Declarations

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