L-Arginine deiminase (ADI) catalyzes the irreversible hydrolysis of L-arginine to citrulline and ammonia. In a previous report of the structure of apoADI from Pseudomonas aeruginosa, the four residues that form the catalytic motif were identified as Cys 406 L-Arginine is used by a number of pathogenic microorganisms to generate ATP via the arginine dihydrolase pathway (1, 2). Arginine deiminase (ADI 2 ; EC 3.5.3.6) ( Fig. 1A) catalyzes the first step of the pathway, wherein arginine is hydrolyzed to citrulline and ammonia. The gene encoding ADI is absent in humans, whereas the enzyme is essential for the survival of pathogenic protozoa and bacteria. Thus, ADI is an attractive antimicrobial drug target candidate. ADI is also a potential anti-angiogenic agent (3) and an antileukemic and nonleukemic murine tumor agent (4).We have previously determined the crystal structure of ADI from Pseudomonas aeruginosa (PaADI) in its unbound state (5). Despite the lack of significant amino acid sequence homology, the core domain structure is similar to those of other arginine-modifying or substituted arginine-modifying enzymes, N ,N -dimethylarginine hydrolase (DDAH) (6), arginine:glycine amidinotransferase (7), arginine:inosamine-phosphate amidinotransferase (8), and human peptidylarginine deiminase (PAD4) (9). Based on the structural similarity and conservation of several key residues in the active site of DDAH and ADI, we proposed a model of arginine binding to ADI in which the guanidinium group is positioned in close proximity to the catalytic Cys 406 (5 ) has also been described (11). The enzyme was co-crystallized with arginine, which gave rise to two different complexes, a tetrahedral adduct and a S-alkylthiouronium adduct. McADI shares 28% sequence identity with PaADI.In the present work, we focused on obtaining the structure of the PaADI⅐L-arginine complex so that we could identify conformational changes that occur upon substrate binding and determine the orientation of substrate binding and catalytic groups in the enzyme⅐substrate complex. The strategy for structure determination employed the C406A mutant, which we had shown in earlier work to be inactive (10). The C406A-arginine structure guided the design of active site mutants in which substrate activation and/or general acid/base catalysis might be impaired. Specifically the H278A, D166A, and D180A mutants were prepared and subjected to kinetic analysis (the results of which are reported in a separate paper) 3 and to crystallization in the presence of L-arginine followed by x-ray structure determination. The structures reported in this paper, are interpreted in the context of the biochemical data to support a model for PaADI substrate recognition and catalysis. ). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The atomic coordinates and structure factors (codes 2a9g, 2aaf, 2abr, and ...

L-Arginine deiminase (ADI) catalyzes the irreversible hydrolysis of arginine to citrulline and ammonia. ADI is involved in the first step of the most widespread anaerobic route of arginine degradation. ADI, missing in high eukaryotes, is a potential antimicrobial and antiparasitic drug target. We have determined the crystal structure of ADI from Pseudomonas aeruginosa by the multiwavelength anomalous diffraction method at 2.45 Å resolution. The structure exhibits similarity to other arginine-modifying or substituted arginine-modifying enzymes such as dimethylarginine dimethylaminohydrolase (DDAH), arginine:glycine amidinotransferase, and arginine:inosamine-phosphate amidinotransferase, despite the lack of significant amino acid sequence homology to these enzymes. The similarity spans a core domain comprising five ␤␤␣␤ motifs arranged in a circle around a 5-fold pseudosymmetry axis. ADI contains an additional ␣-helical domain of novel topology inserted between the first and the second ␤␤␣␤ modules. A catalytic triad, Cys-His-Glu/Asp (arranged in a different manner from that of the thiol proteases), seen in the other arginine-modifying enzymes is also conserved in ADI, as well as many other residues involved in substrate binding. Based on this conservation pattern and the assumption that the substrate binding mode is similar to that of DDAH, an ADI catalytic mechanism is proposed. The main players are Cys-406, which mounts the nucleophilic attack on the carbon atom of the guanidinium group of arginine, and His-278, which serves as a general base.