Insight on the interaction of an agmatinase-like protein with Mn2 + activator ions

• Interactions of an agmatinase-like protein (ALP) with Mn2 + were analyzed.
• Histidine residues were replaced to identify the Mn2 + binding site.
• ALP has one tightly bound Mn2 + and is active in mononuclear form.
• Formation of a binuclear Mn2 + center is promoted at high temperatures.
• Formation of a binuclear Mn2 + center increases both activity and enzyme stability.

Agmatinase is an enzyme that catalyzes the hydrolysis of agmatine, a compound that is associated with numerous functions in the brain of mammalian organisms such as neurotransmitter, anticonvulsant, antinociceptive, anxiolytic and antidepressant-like actions. To date the only characterized agmatinases with significant enzymatic activity were extracted from bacterial organisms. These agmatinases are closely related to another ureahydrolase, arginase; both have binuclear Mn2 + centers in their active sites. An agmatinase-like protein (ALP) from rat brain was identified that bears no sequence homology to known agmatinases (E. Uribe, M. Salas, S. Enriquez, M.S. Orellana, N. Carvajal, Arch. Biochem. Biophys. 461(2007) 146–150). Since all known ureahydrolases contain histidines in their binuclear Mn2 + site each of the five histidine residues in ALP was individually replaced by alanines to identify those that may be involved in metal ion binding. Reactivation assays and thermal stability measurements indicated that His206 is likely to interact with a Mn2 + bound to a high affinity site. In contrast, His65 and possibly His435 are important for binding of a second Mn2 + to a lower affinity site. Metal ion binding to that site is not only leading to an increase in reactivity but also enzyme stability. Thus, similar to bacterial agmatinases and some of the antibiotic-degrading, Zn2 +-dependent metallo-β-lactamases ALP appears to be active in the mono and binuclear form, with binding of the second metal ion increasing both reactivity and stability.

The participation of histidines in Mn2 + interactions in ALP was analyzed. Histidines are not relevant in enzymatic activity of ALP at differences of urea-hydrolase family enzymes, but two histidine participate in Mn2 + interactions. ALP and histidine mutants increase its activity by incubation with Mn2 + at 60 °C, similar to urea-hydrolases enzymes.Figure optionsDownload as PowerPoint slide