Insights into the Mn 2+ Binding Site in the Agmatinase-Like Protein (ALP): A Critical Enzyme for the Regulation of Agmatine Levels in Mammals.

Agmatine is a neurotransmitter with anticonvulsant, anti-neurotoxic and antidepressant-like effects, in addition it has hypoglycemic actions. Agmatine is converted to putrescine and urea by agmatinase (AGM) and by an agmatinase-like protein (ALP), a new type of enzyme which is present in human and rodent brain tissues. Recombinant rat brain ALP is the only mammalian protein that exhibits significant agmatinase activity in vitro and generates putrescine under in vivo conditions. ALP, despite differing in amino acid sequence from all members of the ureohydrolase family, is strictly dependent on Mn ²⁺ for catalytic activity. However, the Mn ²⁺ ligands have not yet been identified due to the lack of structural information coupled with the low sequence identity that ALPs display with known ureohydrolases. In this work, we generated a structural model of the Mn ²⁺ binding site of the ALP and we propose new putative Mn ²⁺ ligands. Then, we cloned and expressed a sequence of 210 amino acids, here called the "central-ALP", which include the putative ligands of Mn ²⁺ . The results suggest that the central-ALP is catalytically active, as agmatinase, with an unaltered K _m for agmatine and a decreased k _cat . Similar to wild-type ALP, central-ALP is activated by Mn ²⁺ with a similar affinity. Besides, a simple mutant D217A, a double mutant E288A/K290A, and a triple mutant N213A/Q215A/D217A of these putative Mn ²⁺ ligands result on the loss of ALP agmatinase activity. Our results indicate that the central-ALP contains the active site for agmatine hydrolysis, as well as that the residues identified are relevant for the ALP catalysis.