Influence of coordination number and ionic radius on metal ion preference and activity of lanthanide-dependent alcohol dehydrogenase: Insights from mutational studies and density functional theory.

Lanthanide (Ln) elements form a cofactor complex with pyrroloquinoline quinone (PQQ) in bacterial alcohol dehydrogenases (Ln-ADH). The lanthanide elements did not support Ln-ADH activity equally, with only early lanthanides (La-Gd) promoting high enzyme activity. However, the early lanthanides did not promote the activity equally and the detailed mechanism of Ln-ADH exhibiting different activity in the presence of different light Lns remains obscure. To uncover the role of lanthanides in promoting Ln-ADH activity, we systemically characterized the activity of an Ln-ADH from Pseudomonas putida KT2440 (PedH) in the presence of various Ln ions. In the results, enzyme activity displayed a bell-shaped trend along with the lanthanide series, with Nd providing the highest activity. Active site mutation analysis revealed that modifying the number of coordinating ligands shifted the metal preference of the enzyme. DFT calculation revealed that the HOMO-LUMO gap, substrate interaction energy and metal ions binding distances were critical for the lanthanides in promoting enzyme activity. This work shed light on the critical role of metal ions in Ln-ADH catalysis, providing insights for future exploration and engineering of Ln-dependent proteins.