Insights into the reaction mechanism of Escherichia coli agmatinase by site‐directed mutagenesis and molecular modelling

Abstract

Upon mutation of Asp153 by asparagine, the catalytic activity of agmatinase (agmatine ureohydrolase, EC 3.5.3.11) from Escherichia coli was reduced to about 5% of wild-type activity. Tryptophan emission fluorescence (k max ¼ 340 nm), and CD spectra were nearly identical for wildtype and D153N agmatinases. The K m value for agmatine (1.6 ± 0.1 mM), as well as the K i for putrescine inhibition (12 ± 2 mM) and the interaction of the enzyme with the required metal ion, were also not altered by mutation. Threedimensional models, generated by homology modelling techniques, indicated that the side chains of Asp153 and Asn153 can perfectly fit in essentially the same position in the active site of E. coli agmatinase. Asp153 is suggested to be involved, by hydrogen bond formation, in the stabilization and orientation of a metal-bound hydroxide for optimal attack on the guanidinium carbon of agmatine. Thus, the disruption of this hydrogen bond is the likely cause of the greately decreased catalytic efficiency of the D153N variant.Keywords: agmatinase; Asp153; site-directed mutagenesis; homology-modelling; E. coli.Agmatinase (agmatine ureohydrolase, EC 3.5.3.11) catalyses the hydrolysis of agmatine to putrescine and urea [1]. Agmatine, which results from decarboxylation of arginine by arginine decarboxylase [2], is a metabolic intermediate in the biosynthesis of putrescine and higher polyamines [1] and may have important regulatory roles in mammals [3][4][5].Agmatinases from Escherichia coli and human tissues, and putative agmatinases from Synechocystic sp. Schizosaccharomyces pombe and Bacillus subtilis, have been cloned and the deduced amino acid sequences indicate their homology to all sequenced arginases [4][5][6][7]; all these enzymes catalyse an hydrolytic reaction with production of urea. The question arises therefore as to whether a similar or identical mechanism is involved in catalysis by these enzymes, which apparently evolved from a single primordial protein [6,7]. In this context, both enzymes exhibit an absolute requirement for Mn 2+ for catalytic activity [8,9]; the well established requirement of a binuclear metal cluster for full catalytic activity of arginase [8] is probably also valid for agmatinase [9]. This is reinforced by the fact that residues known to be metal ligands in arginase are strictly conserved in the sequence of agmatinase [7]. Moreover, a critical role for one conserved histidine residue (His163 in the sequence of E. coli agmatinase) has been shown by chemical modification and site-directed mutagenesis of human and rat liver arginases [10,11] and E. coli agmatinase [12]; similar information was deduced from X-ray crystallographic data for arginase from Bacillus caldovelox [13].Based on the crystal structure of rat liver arginase, it was suggested that arginine hydrolysis involves the participation of a metal-bound hydroxide, which is stabilized for optimal nucleophilic attack at the substrate, by donating an hydrogen bond to Asp128 [8,14,15]. In this connection, the D128G variant of human...

Mutational analysis of substrate recognition by human arginase type I − agmatinase activity of the N130D variant

“…On this basis, our results agree with a rapid equilibrium random release of products from both enzyme variants. The same kinetic mechanism was previously reported for rat liver arginase [21] and also for agmatine hydrolysis by Escherichia coli agmatinase [31].…”

“…The arginase loop (residues 126-143 in the sequence of human liver arginase) contains Asn130 and other residues proposed as ligands for the a-carboxylate group of the substrate arginine. As this is the part of the molecule that makes arginine different from agmatine, our results support the concept that differences in this loop area are key factors in determining the differences in substrate specificity between arginase and agmatinase [31]. Interestingly, when compared with rat liver and B. caldovelox arginases, the active site of Deinococcus radiodurans agmatinase was found to deviate mostly in three regions [33].…”

“…A previously reported homology‐modeled structure of E. coli agmatinase was found to be very similar to those of the crystallographically defined rat liver and B. caldovelox arginases [31] with respect to the number and arrangement of structural elements (α‐helix and β‐sheets). One significant difference was the shorter extension of a loop located at the entrance of the active site cleft.…”

Insights into the interaction of human arginase II with substrate and manganese ions by site‐directed mutagenesis and kinetic studies

“…At this connection, residues known to be involved in binding and hydrolysis of the guanidino group of l ‐arginine by arginase are strictly conserved in the active site of the agmatinases [19]. Moreover, modeling studies have revealed that essentially the same position with respect to the metal ions and conserved catalytically important residues may be adopted by agmatine in E. coli agmatinase and l ‐arginine in B. caldovelox arginase [37], indicating that the substrate specificity of these enzymes rely mainly in substituents at C‐α. This has been, in fact, demonstrated for arginase [38,39] and the same may be safely deduced for agmatinase.…”

Discovery of an operon that participates in agmatine metabolism and regulates biofilm formation in Pseudomonas aeruginosa

“…, 2002). AgDI activity is pivotal in P. aeruginosa polyamine synthesis from arginine because it does not contain the speB product agmatinase that converts agmatine directly into putrescine as in other bacteria (Salas et al. , 2002).…”