Structural Insight into Arginine Degradation by Arginine Deiminase, an Antibacterial and Parasite Drug Target

Galkin, Andrey; Kulakova, Liudmila; Sarıkaya, Elif; Lim, Kian Meng; Howard, Andrew; Herzberg, Osnat

doi:10.1074/jbc.m313410200

Cited by 70 publications

(105 citation statements)

References 30 publications

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“…In agreement with its predicted role as a nucleophile attacking the carbon of the guanidinium moiety, mutation of Cys 365 to serine severely compromised the activity of the enzyme. These observations indicate that AstB uses a catalytic mechanism similar to those of amidinotransferases and deiminases (30,31,36).…”

Section: Resultsmentioning

confidence: 85%

“…Detailed catalytic mechanisms have been proposed for the arginine deaminases (30,31). Surprisingly, comparison of the side chains in the vicinity of the guanidinium moiety of ADI (Protein Data Bank code 1LXY (30)) and AstB shows nearly identical environments (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…When this residue is an aspartate, as in ADI (Asp 161 ), this side chain forms two hydrogen bonds to the guanidinium moiety in the substrate and to the corresponding citrulline atoms in the product. A detailed reaction mechanism for ADI enzymes has been previously proposed (30,31).…”

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confidence: 99%

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Crystal Structure of N-Succinylarginine Dihydrolase AstB, Bound to Substrate and Product, an Enzyme from the Arginine Catabolic Pathway of Escherichia coli

Tocilj¹,

Schrag²,

Li³

et al. 2005

Journal of Biological Chemistry

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The ammonia-producing arginine succinyltransferase pathway is the major pathway in Escherichia coli and related bacteria for arginine catabolism as a sole nitrogen source. This pathway consists of five steps, each catalyzed by a distinct enzyme. Here we report the crystal structure of N-succinylarginine dihydrolase AstB, the second enzyme of the arginine succinyltransferase pathway, providing the first structural insight into enzymes from this pathway. The enzyme exhibits a pseudo 5-fold symmetric ␣/␤ propeller fold of circularly arranged ␤␤␣␤ modules enclosing the active site. The crystal structure indicates clearly that this enzyme belongs to the amidinotransferase (AT) superfamily and that the active site contains a Cys-His-Glu triad characteristic of the AT superfamily. Structures of the complexes of AstB with the reaction product and a C365S mutant with bound the N-succinylarginine substrate suggest a catalytic mechanism that consists of two cycles of hydrolysis and ammonia release, with each cycle utilizing a mechanism similar to that proposed for arginine deiminases. Like other members of the AT superfamily of enzymes, AstB possesses a flexible loop that is disordered in the absence of substrate and assumes an ordered conformation upon substrate binding, shielding the ligand from the bulk solvent, thereby controlling substrate access and product release.

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Section: Resultsmentioning

confidence: 85%

Section: Resultsmentioning

confidence: 99%

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Crystal Structure of N-Succinylarginine Dihydrolase AstB, Bound to Substrate and Product, an Enzyme from the Arginine Catabolic Pathway of Escherichia coli

Tocilj¹,

Schrag²,

Li³

et al. 2005

Journal of Biological Chemistry

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“…Chromatographic materials, buffers, 2-benzyl-2-thiopseudourea, L-arginine, L-citrulline, and agmatine were purchased from Sigma, molecular biological materials were from Invitrogen, [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]-L-arginine (specific activity ) 50 mCi/mmol) was from Moravek Biochemicals, Inc., N ω -amino-L-arginine was from Alexis Biochemicals, and N ω -methyl-L-arginine was from Bachem California Inc. N-Amino-L-citrulline (12) was obtained as a gift from Dr. Bruce King at Wake Forest University.…”

Section: Methodsmentioning

confidence: 99%

Kinetic Analysis of Pseudomonas aeruginosa Arginine Deiminase Mutants and Alternate Substrates Provides Insight into Structural Determinants of Function

Lü

Li²,

Wu³

et al. 2006

Biochemistry

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L-Arginine deiminase from Pseudomonas aeruginosa (PaADI) catalyzes the hydrolysis of arginine to citrulline and ammonia. PaADI belongs to the guanidino group-modifying enzyme superfamily (GMSF), which conserves backbone fold and a Cys-, His-, and Asp-based catalytic core. In this paper the contributions made by the PaADI core residues Cys406, His278, and Asp166 and the contribution from the neighboring Asp280 (conserved in most but not all GMSF members) to catalysis of the formation and hydrolysis of the Cys406-alkyluronium intermediate were accessed by kinetic analysis of site-directed mutants. In addition, solution hydrolysis in a chemical model of the S-alkylthiouronium intermediate was examined to reveal the importance of general base catalysis in the enzymatic reaction. Substitutions of the active site gating residue Arg401, the L-arginine C R NH 3 + (COO -) binding residues, Arg185, Arg243, and Asn160, or the His278 hydrogen bond partner, Glu224, were found to cause dramatic reductions in the enzyme turnover rate. These results are interpreted to suggest that electrostatic interactions play a dominant role in PaADI catalysis. Structural variations observed in P. aeruginosa GMSF enzymes PaADI, agmatine deiminase (PaAgDI), and N ω ,N ω -dimethylarginine dimethylaminohydrolase (PaDDAH) indicate an early divergence of the encoding genes. Arginine analogues that are known substrates for PaAgDI and PaDDAH were tested with PaADI to define clear boundaries of biochemical function in the three hydrolases. The conservation of a catalytic core associated with the common chemical function and the divergence of substrate-binding residues (as well as one key catalytic residue) to expand the substrate range provide insight into the evolution of the catalysts that form the GMSF.The guanidino-modifying enzyme superfamily (hereafter referred to as the GMSF) 1 is a large family of R/ -propellerfold enzymes that catalyze nucleophilic substitution reactions at the guanidinium carbon atom of L-arginine or an L-arginine derivative (for an excellent review, see ref 1). The family is divided into the hydrolase branch, in which an external guanidino NH 2 group is displaced, and the transferase branch, in which the ornithine moiety is displaced. The most remarkable family member, N-succinylarginine dihydrolase (2), catalyzes two consecutive substitution reactions, thus combining the elements of both branches. Aside from N-succinylarginine dihydrolase (3), the genome of Pseudomonas aeruginosa encodes three chemical homologues of the hydrolase branch of the GMSF: arginine deiminase (accession number P13981; locus name PA5171) (PaADI), agmatine deiminase (accession number Q9I6J9; locus name PA0292) (PaAgDI), and N ω ,N ω -dimethylarginine dimethylaminohydrolase (accession number Q9I4E3; locus name PA1195) (PaDDAH). A comparison of the published PaADI (4) and PaDDAH (5) X-ray structures with the recently deposited structure of the AgADI from Arabidopsis thaliana (PDB code 1VKP) (6) demonstrates that despite their low pairwise ...

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“…Our results indicated that 100 mM PDP solution (pH 7.4) containing L-arginine gave higher activity than 10 mM PDP solution (pH 7.4) with 100 mM L-arginine or 100 mM PDP solution (pH 7.4) without L-arginine in the refolding reaction. Since presence of L-arginine in the refolding reaction markedly increased the enzymatic activity, the reaction intermediates between ADI and L-arginine may be tightly bound by the combination of covalent links, polar interactions, or hydrophobic interactions [3,5].…”

Section: Discussionmentioning

confidence: 99%

Growth Inhibition of Human Melanoma Cells by a Recombinant Arginine Deiminase Expressed in Escherichia coli

Kozai

Sasamori

Fujihara

et al. 2009

The Journal of Veterinary Medical Science

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ABSTRACT. We have cloned the arginine deiminase (ADI) gene from Mycoplasma hominis PG21 genomic DNA by polymerase chain reaction, and changed four TGA tryptophan codons (stop codon in E. coli) to TGG codons in the coding region by site-directed mutagenesis in order to express in E. coli. The recombinant ADI (rADI) was purified to apparent homogeneity by Ni-affinity chromatography after extraction from inclusion bodies followed by refolding. The rADI expressed in E. coli was estimated to be 50 kDa. Dimeric forms of rADI exerted enzymatic activity. We found that high concentration of potassium dihydrogenphosphate (PDP) and L-arginine addition in refolding reaction increases the enzyme activity. The specific activity of rADl was calculated as 0.618 U/mg. In addition, the enzyme activity of purified rADI remained for at least one month in 100 mM PDP solution (pH 6.5), but diminished within one week in 100 mM PDP solution (pH 7.4). Anti-tumor activity of the purified rADI was estimated to be 0.036 U/ml as 50% growth inhibitory activity against human melanoma cell line G-361. Arginine dehydrolysing mycoplasmas convert L-arginine into L-citrulline and ammonia by the arginine deiminase (ADI; EC 3.5.3.6) activity [1]. ADI gene of Mycoplasma hominis PG21 has been cloned in Escherichia coli by using plasmid pUC118 [7]. ADI has been shown to affect apoptosis and inhibit NO synthesis (i.e., anti-angiogenic effects), and exert effects against tumor necrosis factor- (TNF-) and neutralize endotoxin. [2,6,11,16,23,27]. ADI has also been known to inhibit the growth of melanoma and hepatocellular carcinoma cells in vitro as well as in vivo [19,20,24,26]. This anti-tumor activity of ADI has been attributed to the depletion of L-arginine, which is essential in these tumor cells [20,25]. Normal cells are not sensitive to external addition of ADI since they produce L-arginine by themselves. Expression of the ADI gene of M. homins PG21 in E. coli has been hampered since the ADI gene of M. hominis has four opal nonsense codons, which are known to encode tryptophan in mycoplasma cells. We have changed these opal codons to universal tryptophan codons and successfully expressed the recombinant ADI (rADI) gene of M. homins in E. coli, and demonstrated the enzymatic activity of purified rADI. MATERIALS AND METHODS Construction of ADI expression vector:Four stop codons TGA in the ADI gene cloned in pUC118 was changed into TGG by site-directed mutagenesis mediated by DpnImethod in order to express in E. coli [21]. Correct introductions of the mutation were confirmed by DNA sequencing. The mutated ADI gene, designated as SYN1903, was recloned into pET-47b (Novagen, Darmstadt, Germany) expression vector.Expression and purification of ADI: Recombinant pET47b plasmids encoding mycoplasmal ADI gene were transformed into competent E. coli BL21 (DE3) cells. The transformant was grown in 600 ml Luria broth (Sigma-Aldrich, St. Louis, MO, U.S.A.) at 30C, and isopropyl 1-thio--Dgalactoside was added at final concentration of 0.1 mM for induction of t...

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Structural Insight into Arginine Degradation by Arginine Deiminase, an Antibacterial and Parasite Drug Target

Cited by 70 publications

References 30 publications

Crystal Structure of N-Succinylarginine Dihydrolase AstB, Bound to Substrate and Product, an Enzyme from the Arginine Catabolic Pathway of Escherichia coli

Crystal Structure of N-Succinylarginine Dihydrolase AstB, Bound to Substrate and Product, an Enzyme from the Arginine Catabolic Pathway of Escherichia coli

Kinetic Analysis of Pseudomonas aeruginosa Arginine Deiminase Mutants and Alternate Substrates Provides Insight into Structural Determinants of Function

Growth Inhibition of Human Melanoma Cells by a Recombinant Arginine Deiminase Expressed in Escherichia coli

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