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.