Crystal structure and molecular dynamics simulation analysis of alanine dehydrogenase from Thermus thermophilus provide insights into its thermostability

The bacterium Thermus thermophilus thrives at high-temperatures, making it a valuable source of heat-stable enzymes for various biotechnological applications. The alanine dehydrogenase (AlaDH) enzyme can catalyze the reversible conversion between alanine and pyruvate. Despite extensive research on AlaDH across various organisms, its three-dimensional structure has only been determined for a few. Here we report the three-dimensional structure of the hexameric AlaDH of T. thermophilus ( Tt AlaDH) solved using the molecular replacement technique. To explore the potential catalytic mechanism of Tt AlaDH, the protein was docked with NAD and pyruvate, and these results were compared with other AlaDH crystal structures with bound substrates. The docking outcomes indicate that the mode of binding of NAD and pyruvate with Tt AlaDH closely resembles those in complex structures like P. lapideum, M. tuberculosis and G. kaustophilus AlaDHs. The molecular dynamics simulations were conducted for Tt AlaDH (thermophilic) and Mt AlaDHs (mesophilic) at three distinct temperatures (300 K, 333 K and 353 K). This study revealed that Tt AlaDH has higher structural stability than Mt AlaDH. Theoretical calculations of the melting temperature (Tm) and folding free energy (ΔG) of different AlaDHs revealed that Tt AlaDH is more prominent than other AlaDHs. The current findings suggest that Tt AlaDH has more potential uses than other bacterial AlaDHs in the production of alanine at high temperatures. L-alanine is used in a wide range of industries, including agriculture, food, and medicine. Researchers can conduct further experiments to modify alanine dehydrogenase to widen its substrate scope beyond its natural predilection for pyruvate with the aid of this Tt AlaDH crystal structure.