Exploring Substituted Dihydroxybenzenes as Urease Inhibitors through Structure–Activity Relationship Studies in Soil Incubations

Urea fertilization as nitrogen (N) source is essential for increasing crop productivity. However, it results in significant N loss through ammonia volatilization, nitrate leaching, and nitrous oxide emissions, causing environmental harm and economic loss. Urease, an enzyme in soil, rapidly catalyzes urea hydrolysis to ammonia/ammonium. To reduce ammonia volatilization, urease inhibitors delay hydrolysis until urea is dissolved in the soil body. The commercial product N-(n-butyl)thiophosphoric triamide (NBPT) is effective only in certain soils, yet it dominates the current global urease inhibitor market. This study examines the performance of un- and substituted dihydroxybenzenes (DHBs) as alternatives to NBPT in two Australian soils. Among them, 4-fluorocatechol (DHB 6) and 4-bromocatechol (DHB 8) are more effective in delaying urea hydrolysis than NBPT in acidic sandy loam soil. Michaelis–Menten kinetics reveals that NBPT acts as a competitive inhibitor, while DHB 8 acts as a noncompetitive inhibitor. Density functional theory calculations suggest that DHB 8 binds to the cysteine residue in the urease mobile flap, reducing its flexibility and preventing it from assisting urea hydrolysis. This study demonstrates the potential of DHBs as efficient urease inhibitors in certain soils, offering an environmentally viable alternative to NBPT.