Exploring the Kinetics and Thermodynamics of a Novel Histidine Ammonia-Lyase from .

Histidine ammonia-lyase (HAL) plays a pivotal role in the non-oxidative deamination of L-histidine to produce -urocanic, a crucial process in amino acid metabolism. This study examines the cloning, purification, and biochemical characterization of a novel HAL from (HAL) and eight active site mutants to assess their effects on substrate binding, catalysis, thermostability, and secondary structure. The HAL enzyme was successfully overexpressed and purified to homogeneity. Its primary sequence displayed 40.7% to 43.7% similarity with other known HALs and shared the same oligomeric structure in solution. Kinetic assays showed that HAL has optimal activity at 85 °C and pH 8.5, with high thermal stability even after preincubation at high temperatures. Mutations at Y52, H82, N194, and E411 resulted in a complete loss of catalytic activity, underscoring their essential role in enzyme function, while mutations at residues Q274, R280, and F325 did not abolish activity but did reduce catalytic efficiency. Notably, mutants R280K and F325Y displayed novel activity with L-histidinamide, expanding the substrate specificity of HAL enzymes. Circular dichroism (CD) analysis showed minor secondary structure changes in the mutants but no significant effect on global HAL folding. These findings suggest that HAL could be a promising candidate for potential biotechnological applications.