Recombinant Expression, Purification, and In Silico Structural Discovery of a Non-TRP-family 5-Hydroxyisourate Hydrolase from Lactiplantibacillus plantarum L123

<title>Abstract</title> <p> 5-Hydroxyisourate hydrolase (HIUHase) has never been identified from any <italic>Lactobacillus</italic> species. This study reports the first purification and structural characterization of a HIUHase from <italic>Lactiplantibacillus plantarum</italic> L123. The recombinant enzyme was expressed in <italic>E. coli</italic> and purified by GST affinity chromatography, yielding a ~ 45 kDa protein with &gt; 95% purity and a specific activity of 30.01 ± 4.90 mU/mg. Bioinformatic analysis revealed that this enzyme lacks the TRP-family domain (PF00576) and belongs to the PF02517 superfamily. AlphaFold2 predicted an all-α-helical fold featuring a putative His-Glu-His catalytic triad, fundamentally distinct from TRP-family enzymes. This finding provides a new structural perspective on HIUHase diversity and extends our understanding of uric acid catabolism in <italic>Lactobacillus</italic> . </p>