Researchers at Northeastern University have made significant progress in understanding the biochemical basis of ornithine transcarbamylase (OTC) deficiency, a rare but severe genetic disorder that disrupts the body’s ability to remove ammonia, a toxic byproduct of protein metabolism. High levels of ammonia can cause brain damage, liver failure, and even death, particularly in newborns, with the most severe form affecting male infants shortly after birth. Led by professors Mary Jo Ondrechen and Penny Beuning, the team developed a novel machine learning tool called Partial Order Optimum Likelihood, or POOL, to predict how specific genetic mutations in the OTC gene impact enzyme function. The OTC enzyme is a key player in the urea cycle, a process that converts nitrogen into urea for safe excretion. When this enzyme is impaired, ammonia accumulates in the bloodstream. The researchers combined POOL with laboratory experiments to analyze 18 mutations linked to OTC deficiency—17 known disease-causing variants and one control. Their approach successfully predicted the functional impact of 17 out of 18 mutations, demonstrating high accuracy. The study, published in ACS Chemical Biology, revealed that some mutations appeared normal in test-tube settings but failed in living cells, highlighting the importance of testing in biologically relevant environments. A key insight came from analyzing a property called μ4, which measures how charged amino acids in the enzyme interact with their surroundings. These interactions are essential for the enzyme’s ability to catalyze reactions. The team found that mutations disrupting these electrostatic interactions were more likely to impair enzyme function. Importantly, the study uncovered that not all disease-linked mutations directly interfere with the enzyme’s catalytic activity. Some may affect protein stability, cellular production, or interactions with other proteins in the metabolic pathway—questions that remain under investigation. With around 14,000 to 77,000 individuals diagnosed annually worldwide, OTC deficiency currently has no cure. Treatments include strict low-protein diets, medications to remove excess nitrogen, and in severe cases, liver transplants. The new findings open the door to more targeted therapies, such as small molecules designed to stabilize or restore function in mutant enzymes. The research was made possible by the dedicated work of Northeastern’s doctoral students, who produced and tested the mutant enzymes in the lab. The team’s success underscores the power of combining machine learning with experimental biochemistry to decode complex genetic diseases. Ondrechen and Beuning now aim to uncover the underlying mechanisms behind mutations that don’t directly affect catalysis, a critical step toward developing personalized treatments. Their work represents a major leap in understanding how genetic variation translates into disease at the molecular level.