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Human cytochrome P450 (P450) 27A1 catalyzes the hydroxylation of cholesterol and vitamin D derivatives. P450 27A1 is localized in the mitochondria and is reduced by its redox partner protein adrenodoxin twice for each catalytic cycle. The reliance on adrenodoxin is conserved across all human mitochondrial P450 enzymes. This study examines the adrenodoxin interaction with P450 27A1 and draws comparisons with studies of other P450 enzymes to determine if differences exist. The P450-adrenodoxin complex structure was examined by chemical crosslinking and analyzed by mass spectrometry. The effect of adrenodoxin concentration on P450 27A1 function was assessed by studying effects on steady state enzyme kinetics parameters and equilibrium substrate binding. The results suggest that adrenodoxin binds to P450 27A1 at a proximal site like other P450 enzymes but differs in the specific residues involved. Furthermore, the presence of adrenodoxin and/or substrate decreases the number of interprotein and intraprotein crosslinks observed, indicating that these components change the conformation of the P450 enzyme. Increased adrenodoxin concentration causes the P450 and vitamin D3 kcat value to increase, the kcat/Km value to decrease, and the substrate Kd to remain constant. These results suggest adrenodoxin alters enzyme efficiency beyond electron transfer without affecting substrate loading. The adrenodoxin effects on P450 27A1 kinetics and equilibrium constants differ from those of other human mitochondrial P450 enzymes. In total, these structural and functional studies suggest that while the general adrenodoxin binding site and function is conserved across P450 enzymes, the details and additional effects of this interaction vary. 

GM1 gangliosidosis (GM1) is a rare but fatal neurodegenerative disease caused by dysfunction or lack of production of lysosomal enzyme, β-galactosidase, leading to accumulation of substrates. The most promising treatments for GM1, include enzyme replacement therapy (ERT), substrate reduction therapy (SRT), stem cell therapy and gene editing. However, effectiveness is limited for neuropathic GM1 due to the restrictive nature of the blood–brain barrier (BBB). ERT and SRT alleviate substrate accumulation through exogenous supplementation over the patient’s lifetime, while gene editing could be curative, fixing the causative gene,GLB1, to enable endogenous enzyme activity. Stem cell therapy can be a combination of both, withex vivogene editing of cells to cause the production of enzymes. These approaches require special considerations for brain delivery, which has led to novel formulations. A few therapeutic interventions have progressed to early-phase clinical trials, presenting a bright outlook for improved clinical management for GM1.