Crystal Structure of Escherichia coli Agmatinase: Catalytic Mechanism and Residues Relevant for Substrate Specificity

Abstract

Agmatine is the product of the decarboxylation of L-arginine by the enzyme arginine decarboxylase. This amine has been attributed to neurotransmitter functions, anticonvulsant, anti-neurotoxic, and antidepressant in mammals and is a potential therapeutic agent for diseases such as Alzheimer’s, Parkinson’s, and cancer. Agmatinase enzyme hydrolyze agmatine into urea and putrescine, which belong to one of the pathways producing polyamines, essential for cell proliferation. Agmatinase from Escherichia coli (EcAGM) has been widely studied and kinetically characterized, described as highly specific for agmatine. In this study, we analyze the amino acids involved in the high specificity of EcAGM, performing a series of mutations in two loops critical to the active-site entrance. Two structures in different space groups were solved by X-ray crystallography, one at low resolution (3.2 Å), including a guanidine group; and other at high resolution (1.8 Å) which presents urea and agmatine in the active site. These structures made it possible to understand the interface interactions between subunits that allow the hexameric state and postulate a catalytic mechanism according to the Mn2+ and urea/guanidine binding site. Molecular dynamics simulations evaluated the conformational dynamics of EcAGM and residues participating in non-binding interactions. Simulations showed the high dynamics of loops of the active site entrance and evidenced the relevance of Trp68, located in the adjacent subunit, to stabilize the amino group of agmatine by cation-pi interaction. These results allow to have a structural view of the best-kinetic characterized agmatinase in literature up to now.

New Insights into the Determinants of Specificity in Human Type I Arginase: Generation of a Mutant That Is Only Active with Agmatine as Substrate

“…When analyzing the available crystallographic structures for arginase [ 5 , 26 , 27 , 28 , 29 , 30 ] and agmatinase [ 20 , 21 , 31 ], the main differences in their structures lie in two loops located at the entrance to the active site. The ligands for the α-carboxyl group of arginine are located in loop A ; in this loop the residues N130, S137 and N139 stabilize the alpha carboxyl group of arginine, either by direct hydrogen bonding or with a molecule of water ( Figure 2 B).…”

“…As already mentioned, another of the major structural differences between arginase and agmatinase lies in a loop located at the entrance of the active site of both enzymes, which we have identified as loop B ( Figure 2 A). According to crystallographic structures available for arginase [ 5 , 26 , 27 , 28 , 29 , 30 ] and agmatinase [ 20 , 21 , 31 ] in the case of human liver arginase, this loop includes four residues; from them two residues stand out of aspartate that would interact with the alpha-amino group of arginine ( Figure 2 B).…”

“…Enzymes belonging to this arginase family catalyze hydrolysis reactions with the production of urea or formamide as one of the products. Although it is accepted that these enzymes evolved from a common ancestor and subsequently acquired their substrate specificity [ 13 ], so far there are few studies on the molecular basis of such marked differences in specificity [ 17 , 18 , 19 , 20 , 21 ]. In this sense, arginase and agmatinase, despite the close structural analogies between its substrates and the equivalent functions that have been assigned to residues related to catalysis and metal binding, arginine is not a substrate for agmatinase and agmatine is practically not hydrolyzed by arginase.…”

“…By superposition of the structures from human liver arginase, D. radiodurans, and E. coli agmatinase [ 21 , 25 ] ( Figure 2 A), the overall fold and topology of the enzyme is maintained, in the arrangement and quantity of α-helical and β-sheets structures. Furthermore, the position of the Mn 2+ and the residues that bind the guanidine group from the substrate are conserved in these structures.…”

“…This loop B includes the residues D181-P184, which interact with the alpha amino group of arginine ( Figure 2 B). In the structure of agmatinase, this loop appears more open than arginase [ 21 ].…”