Mechanisms of catalysis and inhibition operative in the arginine deiminase from the human pathogen Giardia lamblia

Abstract

Giardia lamblia arginine deiminase (GlAD), the topic of this paper, belongs to the hydrolase branch of the guanidine-modifying enzyme superfamily, whose members employ Cys-mediated nucleophilic catalysis to promote deimination of L-arginine and its naturally occurring derivatives. G. lamblia is the causative agent in the human disease giardiasis. The results of RNAi/antisense RNA gene-silencing studies reported herein indicate that GlAD is essential for G. lamblia trophozoite survival and thus, a potential target for the development of therapeutic agents for the treatment of giardiasis. The homodimeric recombinant protein was prepared in E. coli for in-depth biochemical characterization. The 2-domain GlAD monomer consists of a N-terminal domain that shares an active site structure (depicted by an in-silico model) and kinetic properties (determined by steady-state and transient state kinetic analysis) with its bacterial AD counterparts, and a C-terminal domain of unknown fold and function. GlAD was found to be active over a wide pH range and to accept L-arginine, L-arginine ethyl ester, Nα-benzoyl-L-arginine, and Nω-amino-L-arginine as substrates but not agmatine, L-homoarginine, Nα-benzoyl-L-arginine ethyl ester or a variety of arginine-containing peptides. The intermediacy of a Cys424-alkylthiouronium ion covalent enzyme adduct was demonstrated and the rate constants for formation (k1= 80 s−1) and hydrolysis (k2 = 35 s−1) of the intermediate were determined. The comparatively lower value of the steady state rate constant (kcat = 2.6 s−1), suggests that a step following citrulline formation is rate-limiting. Inhibition of GlAD using Cys directed agents was briefly explored. S-nitroso-L-homocysteine was shown to be an active site directed, irreversible inhibitor whereas Nω-cyano-L-arginine did not inhibit GlAD but instead proved to be an active site directed, irreversible inhibitor of the Bacillus cereus AD.

Plasmid Vectors for Proteomic Analyses in Giardia: Purification of Virulence Factors and Analysis of the Proteasome

“…OCT is known to be immunogenic in humans (31), but it did not induce any antibody response in immunized mice, nor did it confer protection (13). ADI could not be expressed successfully in the Salmonella vaccine strain and remains unexplored as a vaccine candidate (13), but it has been promoted as a drug candidate for therapeutic treatment of giardiasis due to the essentiality of ADI in trophozoites (25). Therefore, we aimed to answer the question of whether both Giardia proteins could be produced in and purified from the parasite, allowing subsequent interaction experiments with human epithelial cell lines in vitro, drug screenings, and vaccine trials in vivo.…”

“…Arginine, moreover, is the preferred substrate for energy production via the arginine dihydrolase (ADH) pathway that is usually found in prokaryotes (20). The secretion of two enzymes of this pathway, arginine deiminase (ADI) and ornithine carbamoyl transferase (OCT), by the parasite has recently been shown to be increased upon interaction with human cells (33), and they have also been suggested to be good drug targets (25). The possibility of producing potential virulence factors as recombinant proteins with native characteristics could further promote research on how Giardia subverts the intestinal milieu to its own benefit and will be valuable in drug screening.…”

Giardia duodenalis Arginine Deiminase Modulates the Phenotype and Cytokine Secretion of Human Dendritic Cells by Depletion of Arginine and Formation of Ammonia

“…We cloned the adi gene present in strain WB-C6 and produced the respective recombinant enzyme, as well as an enzymatically inactive mutant form, ADI C424A (23), in which Cys at residue 424 was replaced with Ala, as hexahistidine-tagged proteins in E. coli. The recombinant proteins were purified, and the wild-type protein had an ADI activity level of 6.8 U/mg.…”

Insights into the structure and inhibition ofGiardia intestinalisarginine deiminase: homology modeling, docking, and molecular dynamics studies

“…Nevertheless, the fold and function of this domain have not been elucidated. Previous reports have identified GiADI as a homodimer and have shown that the Cterminal domain is not involved in dimer interface formation (Knodler, Schofield, Gooley, & Edwards, 1997;Li et al, 2009). Based on this precedent, the 145 amino acids of the C-terminal domain were omitted during the modeling construction.…”

“…Since GiADI has been found in experimental studies as a homodimer enzyme (Li et al, 2009), the quaternary structure was constructed with the model selected above. For this purpose, the crystallographic structure of M. arginini ADI (Das et al, 2004) was used as a template to determine the three-dimensional arrangement of the homodimer.…”