Insight into the functional and structural relationship of ornithine decarboxylase and its mutants from Lacticaseibacillus rhamnosus

Ornithine decarboxylase (ODC) is a critical enzyme in the polyamine biosynthesis pathway that catalyzes the conversion of l-ornithine to putrescine using pyridoxal 5′-phosphate (PLP). Lactobacilli act probiotically by stimulating the immune system, defending against pathogens, and mitigating the impact of various chronic illnesses. To better understand the function and structure of ODC from Lacticaseibacillus rhamnosus (LrODC-WT), we investigated its enzymatic activity, kinetic characteristics, crystal structure, and further examined a variety of single-residue mutants. We found that active LrODC-WT has the following kinetic parameters: K_M 6.83 ± 1.01 mM, k_cat 1.44 ± 0.1 s⁻¹, and k_cat/K_M 210.83 ± 19.37 M⁻¹ s⁻¹. Unlike LrODC-WT, H346A, F186A, H216F, and E281Q showed no catalytic activity. H346A showed maximum unfolding at 2 M guanidine hydrochloride (GdnHCl), while the other enzymes exhibited peak unfolding effects at 1 M, indicating that H346A shows higher resistance to GdnHCl. The PLP binding in LrODCs using a UV/Vis spectrometer showed that LrODC-WT possesses high PLP, while F186A and H346A demonstrated 50% PLP of LrODC-WT, and absent in H216F and E281Q. The crystal structure of LrODC-WT was identified as a tetramer in which PLP was bound to all four subunits and interacted with residue K347 for Schiff base formation. While the crystal structures of H216F and H346A form dimers, an LrODC-WT tetramer can form via hydrogen bonding of D544 and N270. An improved understanding of the structure and function of LrODCs is relevant for controlling its polyamine production and for optimizing L. rhamnosus strains for use as a more potent probiotic.