Spectroscopic studies on the [4Fe-4S] cluster in adenosine 5'-phosphosulfate reductase from Mycobacterium tuberculosis

Mycobacterium tuberculosis adenosine 5'-phosphosulfate reductase (MtAPR) is an iron-sulfur protein and a validated target to develop new antitubercular agents, particularly for the treatment of latent infection. The enzyme harbors a [4Fe-4S](2+) cluster that is coordinated by four cysteinyl ligands, two of which are adjacent in the amino acid sequence. The iron-sulfur cluster is essential for catalysis; however, the precise role of the [4Fe-4S] cluster in APR remains unknown. Progress in this area has been hampered by the failure to generate a paramagnetic state of the [4Fe-4S] cluster that can be studied by electron paramagnetic resonance spectroscopy. Herein, we overcome this limitation and report the EPR spectra of MtAPR in the [4Fe-4S](+) state. The EPR signal is rhombic and consists of two overlapping S = ½ species. Substrate binding to MtAPR led to a marked increase in the intensity and resolution of the EPR signal and to minor shifts in principle g values that were not observed among a panel of substrate analogs, including adenosine 5'-diphosphate. Using site-directed mutagenesis, in conjunction with kinetic and EPR studies, we have also identified an essential role for the active site residue Lys-144, whose side chain interacts with both the iron-sulfur cluster and the sulfate group of adenosine 5'-phosphosulfate. The implications of these findings are discussed with respect to the role of the iron-sulfur cluster in the catalytic mechanism of APR.     

Characterisation of a siderophore from Acinetobacter calcoaceticus

Abstract The Gram-negatice bacterium Acinetobacter calcoaceticus was examined for production of siderophores and iron-repressible outer membrane proteins following growth in iron-restricted media. The iron chelator, 2,3-dihydroxybenzoic acid was identified in the culture supernatant bu ¹H nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). A group of outer membrane proteins between 80 and 85 kDa were induced under iron restriction.     

Identification and characterization of oxalate oxidoreductase, a novel thiamine pyrophosphate-dependent 2-oxoacid oxidoreductase that enables anaerobic growth on oxalate

Moorella thermoacetica is an anaerobic acetogen, a class of bacteria that is found in the soil, the animal gastrointestinal tract, and the rumen. This organism engages the Wood-Ljungdahl pathway of anaerobic CO(2) fixation for heterotrophic or autotrophic growth. This paper describes a novel enzyme, oxalate oxidoreductase (OOR), that enables M. thermoacetica to grow on oxalate, which is produced in soil and is a common component of kidney stones. Exposure to oxalate leads to the induction of three proteins that are subunits of OOR, which oxidizes oxalate coupled to the production of two electrons and CO(2) or bicarbonate. Like other members of the 2-oxoacid:ferredoxin oxidoreductase family, OOR contains thiamine pyrophosphate and three [Fe(4)S(4)] clusters. However, unlike previously characterized members of this family, OOR does not use coenzyme A as a substrate. Oxalate is oxidized with a k(cat) of 0.09 s(-1) and a K(m) of 58 μM at pH 8. OOR also oxidizes a few other 2-oxoacids (which do not induce OOR) also without any requirement for CoA. The enzyme transfers its reducing equivalents to a broad range of electron acceptors, including ferredoxin and the nickel-dependent carbon monoxide dehydrogenase. In conjunction with the well characterized Wood-Ljungdahl pathway, OOR should be sufficient for oxalate metabolism by M. thermoacetica, and it constitutes a novel pathway for oxalate metabolism.     

Certhrax Toxin,an Anthrax-related ADP-ribosyltransferase from Bacillus cereus

We identified Certhrax, the first anthrax-like mART toxin from the pathogenic G9241 strain of Bacillus cereus. Certhrax shares 31% sequence identity with anthrax lethal factor from Bacillus anthracis; however, we have shown that the toxicity of Certhrax resides in the mART domain, whereas anthrax uses a metalloprotease mechanism. Like anthrax lethal factor, Certhrax was found to require protective antigen for host cell entry. This two-domain enzyme was shown to be 60-fold more toxic to mammalian cells than anthrax lethal factor. Certhrax localizes to distinct regions within mouse RAW264.7 cells by 10 min postinfection and is extranuclear in its cellular location. Substitution of catalytic residues shows that the mART function is responsible for the toxicity, and it binds NAD⁺ with high affinity (K_D = 52.3 ± 12.2 μm). We report the 2.2 Å Certhrax structure, highlighting its structural similarities and differences with anthrax lethal factor. We also determined the crystal structures of two good inhibitors (P6 (K_D = 1.7 ± 0.2 μm, K_i = 1.8 ± 0.4 μm) and PJ34 (K_D = 5.8 ± 2.6 μm, K_i = 9.6 ± 0.3 μm)) in complex with Certhrax. As with other toxins in this family, the phosphate-nicotinamide loop moves toward the NAD⁺ binding site with bound inhibitor. These results indicate that Certhrax may be important in the pathogenesis of B. cereus.     

Isolation and partial characterization of phenolate siderophore from Rhizobium leguminosarum IARI 102

Abstract Rhizobium leguminosarum IARI 102 produced 2,3-dihydroxy benzoic acid, a type of phenolate siderophore, under iron-starved conditions. Hydroxamic acids were not detected. Maximum production of siderophore was found at 26 h of growth in a chemically defined medium at 28°C with shaking. Threonine was detected as the amino acid conjugate of the siderophore. Addition of Fe³⁺ to the culture medium increased the growth yield significantly, but depressed the production of the iron chelating compound.     

Effects of freshwater bacterial siderophore on Microcystis and Anabaena

Six siderophore-producing bacterial strains were isolated from the freshwater, in which five strains belonged to Pseudomona genus, and the other belonged to Stenotrophomonas genus. The strain, Stenotrophomonas maltophilia 15, which produced hydroxamate-type siderophore, was selected for siderophore preparation. Its siderophore production was inhibited by FeCl₃, especially when FeCl₃ concentration was higher than 20 μM. Effects of siderophore on cyanobacteria Microcystis aeruginosa FACHB-905 and Anabaena flos-aquae FACHB-245 were studied. Compared to the control, almost all the treated groups showed decrease in growth rate and chlorophyll a, carotenoids, phycocyanin, soluble protein, microcystin content, which was attributed the low iron bioavailability in the culture medium. In the study, S. maltophilia 15 showed algicidal activities by secreting siderophore and could inhibit cyanobacterial growth, especially when iron bioavailabity is very low. The two cyanobacterial strains showed distinct demand for iron. It was deduced that in the freshwater the competition between bacteria and cyanobacteria existed for the low-bioavailable iron, which may relate to the replacement of dominant cyanobacteria.     

The C-terminal domain of 4-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii is an autoinhibitory domain

p-Hydroxyphenylacetate (HPA) 3-hydroxylase from Acinetobacter baumannii consists of a reductase component (C(1)) and an oxygenase component (C(2)). C(1) catalyzes the reduction of FMN by NADH to provide FMNH(-) as a substrate for C(2). The rate of reduction of flavin is enhanced ～20-fold by binding HPA. The N-terminal domain of C(1) is homologous to other flavin reductases, whereas the C-terminal domain (residues 192-315) is similar to MarR, a repressor protein involved in bacterial antibiotic resistance. In this study, three forms of truncated C(1) variants and single site mutation variants of residues Arg-21, Phe-216, Arg-217, Ile-246, and Arg-247 were constructed to investigate the role of the C-terminal domain in regulating C(1). In the absence of HPA, the C(1) variant in which residues 179-315 were removed (t178C(1)) was reduced by NADH and released FMNH(-) at the same rates as wild-type enzyme carries out these functions in the presence of HPA. In contrast, variants with residues 231-315 removed behaved similarly to the wild-type enzyme. Thus, residues 179-230 are involved in repressing the production of FMNH(-) in the absence of HPA. These results are consistent with the C-terminal domain in the wild-type enzyme being an autoinhibitory domain that upon binding the effector HPA undergoes conformational changes to allow faster flavin reduction and release. Most of the single site variants investigated had catalytic properties similar to those of the wild-type enzyme except for the F216A variant, which had a rate of reduction that was not stimulated by HPA. F216A could be involved with HPA binding or in the required conformational change for stimulation of flavin reduction by HPA.     

Deciphering the role of histidine 252 in mycobacterial adenosine 5'-phosphosulfate (APS) reductase catalysis

Mycobacterium tuberculosis adenosine 5'-phosphosulfate reductase (APR) catalyzes the first committed step in sulfate reduction for the biosynthesis of cysteine and is essential for survival in the latent phase of tuberculosis infection. The reaction catalyzed by APR involves the nucleophilic attack by conserved Cys-249 on adenosine 5'-phosphosulfate, resulting in a covalent S-sulfocysteine intermediate that is reduced in subsequent steps by thioredoxin to yield the sulfite product. Cys-249 resides on a mobile active site lid at the C terminus, within a K(R/T)ECG(L/I)H motif. Owing to its strict conservation among sulfonucleotide reductases and its proximity to the active site cysteine, it has been suggested that His-252 plays a key role in APR catalysis, specifically as a general base to deprotonate Cys-249. Using site-directed mutagenesis, we have changed His-252 to an alanine residue and analyzed the effect of this mutation on the kinetic parameters, pH rate profile, and ionization of Cys-249 of APR. Interestingly, our data demonstrate that His-252 does not perturb the pK(a) of Cys-249 or play a direct role in rate-limiting chemical steps of the reaction. Rather, we show that His-252 enhances substrate affinity via interaction with the α-phosphate and the endocyclic ribose oxygen. These findings were further supported by isothermal titration calorimetry to provide a thermodynamic profile of ligand-protein interactions. From an applied standpoint, our study suggests that small-molecules targeting residues in the dynamic C-terminal segment, particularly His-252, may lead to inhibitors with improved binding affinity.     

Characterization of a novel sesquiterpene cyclase involved in (+)-caryolan-1-ol biosynthesis in Streptomyces griseus

Most terpenoids have been isolated from plants and fungi and only a few from bacteria. However, an increasing number of genome sequences indicate that bacteria possess a variety of terpenoid cyclase genes. We characterized a sesquiterpene cyclase gene (SGR2079, named gcoA) found in Streptomyces griseus. When expressed in Streptomyces lividans, gcoA directed production of a sesquiterpene, isolated and determined to be (+)-caryolan-1-ol using spectroscopic analyses. (+)-Caryolan-1-ol was also detected in the crude cell lysate of wild-type S. griseus but not in a gcoA knockout mutant, indicating that GcoA is a genuine (+)-caryolan-1-ol synthase. Enzymatic properties were characterized using N-terminally histidine-tagged GcoA, produced in Escherichia coli. As expected, incubation of the recombinant GcoA protein with farnesyl diphosphate yielded (+)-caryolan-1-ol. However, a small amount of another sesquiterpene was also detected. This was identified as the bicyclic sesquiterpene hydrocarbon (+)-β-caryophyllene by comparison with an authentic sample using GC-MS. Incorporation of a deuterium atom into the C-9 methylene of (+)-caryolan-1-ol in an in vitro GcoA reaction in deuterium oxide indicated that (+)-caryolan-1-ol was synthesized by a proton attack on the C-8/C-9 double bond of (+)-β-caryophyllene. Several β-caryophyllene synthases have been identified from plants, but these cannot synthesize caryolan-1-ol. Although caryolan-1-ol has been isolated previously from several plants, the enzyme responsible for its biosynthesis has not been identified previously. GcoA is thus the first known caryolan-1-ol synthase. Isolation of caryolan-1-ol from microorganisms is unprecedented.     

Mercaptosuccinate Dioxygenase,a Cysteine Dioxygenase Homologue,from Variovorax paradoxus Strain B4 Is the Key Enzyme of Mercaptosuccinate Degradation

The versatile thiol mercaptosuccinate has a wide range of applications, e.g. in quantum dot research or in bioimaging. Its metabolism is investigated in Variovorax paradoxus strain B4, which can utilize this compound as the sole source of carbon and sulfur. Proteomic studies of strain B4 resulted in the identification of a putative mercaptosuccinate dioxygenase, a cysteine dioxygenase homologue, possibly representing the key enzyme in the degradation of mercaptosuccinate. Therefore, the putative mercaptosuccinate dioxygenase was heterologously expressed, purified, and characterized in this study. The results clearly demonstrated that the enzyme utilizes mercaptosuccinate with concomitant consumption of oxygen. Thus, the enzyme is designated as mercaptosuccinate dioxygenase. Succinate and sulfite were verified as the final reaction products. The enzyme showed an apparent K_m of 0.4 mm, and a specific activity (V_max) of 20.0 μmol min⁻¹ mg⁻¹ corresponding to a k_cat of 7.7 s⁻¹. Furthermore, the enzyme was highly specific for mercaptosuccinate, no activity was observed with cysteine, dithiothreitol, 2-mercaptoethanol, and 3-mercaptopropionate. These structurally related thiols did not have an inhibitory effect either. Fe(II) could clearly be identified as metal cofactor of the mercaptosuccinate dioxygenase with a content of 0.6 mol of Fe(II)/mol of enzyme. The recently proposed hypothesis for the degradation pathway of mercaptosuccinate based on proteome analyses could be strengthened in the present study. (i) Mercaptosuccinate is first converted to sulfinosuccinate by this mercaptosuccinate dioxygenase; (ii) sulfinosuccinate is spontaneously desulfinated to succinate and sulfite; and (iii) whereas succinate enters the central metabolism, sulfite is detoxified by the previously identified putative molybdopterin oxidoreductase.     

Functional and structural analysis of the siderophore synthetase AsbB through reconstitution of the petrobactin biosynthetic pathway from Bacillus anthracis

Petrobactin, a mixed catechol-carboxylate siderophore, is required for full virulence of Bacillus anthracis, the causative agent of anthrax. The asbABCDEF operon encodes the biosynthetic machinery for this secondary metabolite. Here, we show that the function of five gene products encoded by the asb operon is necessary and sufficient for conversion of endogenous precursors to petrobactin using an in vitro system. In this pathway, the siderophore synthetase AsbB catalyzes formation of amide bonds crucial for petrobactin assembly through use of biosynthetic intermediates, as opposed to primary metabolites, as carboxylate donors. In solving the crystal structure of the B. anthracis siderophore biosynthesis protein B (AsbB), we disclose a three-dimensional model of a nonribosomal peptide synthetase-independent siderophore (NIS) synthetase. Structural characteristics provide new insight into how this bifunctional condensing enzyme can bind and adenylate multiple citrate-containing substrates followed by incorporation of both natural and unnatural polyamine nucleophiles. This activity enables formation of multiple end-stage products leading to final assembly of petrobactin. Subsequent enzymatic assays with the nonribosomal peptide synthetase-like AsbC, AsbD, and AsbE polypeptides show that the alternative products of AsbB are further converted to petrobactin, verifying previously proposed convergent routes to formation of this siderophore. These studies identify potential therapeutic targets to halt deadly infections caused by B. anthracis and other pathogenic bacteria and suggest new avenues for the chemoenzymatic synthesis of novel compounds.     

Isolation and partial characterization of a compound with siderophore activity from Vibrio parahaemolyticus

A compound with siderophore activity was purified by successive column and thin layer chromatographic procedures from Dowex 1 x 8 extracts of culture supernatants of Vibrio parahaemolyticus AQ 3354. The strain synthesized the compound in culture media containing less than 2 microM added FeCl3. Hydrolysis of the compound yielded alanine, ethanolamine, citric acid and 2-ketoglutaric acid. The 1H-NMR spectrum exhibited the presence of a residue from each of these components in the intact molecule. The fast-atom bombardment mass spectrum of the methyl ester derivative indicated a prominent ion at m/z 477, probably corresponding to [M + 1] ion. Other strains of V. parahaemolyticus were also found to produce this compound when grown in an iron-limited medium.     

Dissecting the metabolic roles of pteridine reductase 1 in Trypanosoma brucei and Leishmania major

Leishmania parasites are pteridine auxotrophs that use an NADPH-dependent pteridine reductase 1 (PTR1) and NADH-dependent quinonoid dihydropteridine reductase (QDPR) to salvage and maintain intracellular pools of tetrahydrobiopterin (H(4)B). However, the African trypanosome lacks a credible candidate QDPR in its genome despite maintaining apparent QDPR activity. Here we provide evidence that the NADH-dependent activity previously reported by others is an assay artifact. Using an HPLC-based enzyme assay, we demonstrate that there is an NADPH-dependent QDPR activity associated with both TbPTR1 and LmPTR1. The kinetic properties of recombinant PTR1s are reported at physiological pH and ionic strength and compared with LmQDPR. Specificity constants (k(cat)/K(m)) for LmPTR1 are similar with dihydrobiopterin (H(2)B) and quinonoid dihydrobiopterin (qH(2)B) as substrates and about 20-fold lower than LmQDPR with qH(2)B. In contrast, TbPTR1 shows a 10-fold higher k(cat)/K(m) for H(2)B over qH(2)B. Analysis of Trypanosoma brucei isolated from infected rats revealed that H(4)B (430 nM, 98% of total biopterin) was the predominant intracellular pterin, consistent with a dual role in the salvage and regeneration of H(4)B. Gene knock-out experiments confirmed this: PTR1-nulls could only be obtained from lines overexpressing LmQDPR with H(4)B as a medium supplement. These cells grew normally with H(4)B, which spontaneously oxidizes to qH(2)B, but were unable to survive in the absence of pterin or with either biopterin or H(2)B in the medium. These findings establish that PTR1 has an essential and dual role in pterin metabolism in African trypanosomes and underline its potential as a drug target.     

Identification of an Active Site-bound Nitrile Hydratase Intermediate through Single Turnover Stopped-flow Spectroscopy

Stopped-flow kinetic data were obtained for the iron-type nitrile hydratase from Rhodococcus equi TG328-2 (ReNHase) using methacrylonitrile as the substrate. Multiple turnover experiments suggest a three-step kinetic model that allows for the reversible binding of substrate, the presence of an intermediate, and the formation of product. Microscopic rate constants determined from these data are in good agreement with steady state data confirming that the stopped-flow method used was appropriate for the reaction. Single turnover stopped-flow experiments were used to identify catalytic intermediates. These data were globally fit confirming a three-step kinetic model. Independent absorption spectra acquired between 0.005 and 0.5 s of the reaction reveal a significant increase in absorbance at 375, 460, and 550 nm along with the hypsochromic shift of an Fe³⁺←S ligand-to-metal charge transfer band from 700 to 650 nm. The observed UV-visible absorption bands for the Fe³⁺-nitrile intermediate species are similar to low spin Fe³⁺-enzyme and model complexes bound by NO or N₃⁻. These data provide spectroscopic evidence for the direct coordination of the nitrile substrate to the nitrile hydratase active site low spin Fe³⁺ center.     

Identification through combinatorial random and rational mutagenesis of a substrate-interacting exosite in the gamma domain of streptokinase

To identify new structure-function correlations in the γ domain of streptokinase, mutants were generated by error-prone random mutagenesis of the γ domain and its adjoining region in the β domain followed by functional screening specifically for substrate plasminogen activation. Single-site mutants derived from various multipoint mutation clusters identified the importance of discrete residues in the γ domain that are important for substrate processing. Among the various residues, aspartate at position 328 was identified as critical for substrate human plasminogen activation through extensive mutagenesis of its side chain, namely D328R, D328H, D328N, and D328A. Other mutants found to be important in substrate plasminogen activation were, namely, R319H, N339S, K334A, K334E, and L335Q. When examined for their 1:1 interaction with human plasmin, these mutants were found to retain the native-like high affinity for plasmin and also to generate amidolytic activity with partner plasminogen in a manner similar to wild type streptokinase. Moreover, cofactor activities of the mutants precomplexed with plasmin against microplasminogen as the substrate as well as in silico modeling studies suggested that the region 315-340 of the γ domain interacts with the serine protease domain of the macromolecular substrate. Overall, our results identify the presence of a substrate specific exosite in the γ domain of streptokinase.     

Analysis of keystone enzyme in Agar hydrolysis provides insight into the degradation (of a polysaccharide from) red seaweeds

Agars are abundant polysaccharides from marine red algae, and their chemical structure consists of alternating D-galactose and 3,6-anhydro-L-galactose residues, the latter of which are presumed to make the polymer recalcitrant to degradation by most terrestrial bacteria. Here we study a family 117 glycoside hydrolase (BpGH117) encoded within a recently discovered locus from the human gut bacterium Bacteroides plebeius. Consistent with this locus being involved in agarocolloid degradation, we show that BpGH117 is an exo-acting 3,6-anhydro-α-(1,3)-L-galactosidase that removes the 3,6-anhydrogalactose from the non-reducing end of neoagaro-oligosaccharides. A Michaelis complex of BpGH117 with neoagarobiose reveals the distortion of the constrained 3,6-anhydro-L-galactose into a conformation that favors catalysis. Furthermore, this complex, supported by analysis of site-directed mutants, provides evidence for an organization of the active site and positioning of the catalytic residues that are consistent with an inverting mechanism of catalysis and suggests that a histidine residue acts as the general acid. This latter feature differs from the vast majority of glycoside hydrolases, which use a carboxylic acid, highlighting the alternative strategies that enzymes may utilize in catalyzing the cleavage of glycosidic bonds.     

Functional characterization of the promiscuous prenyltransferase responsible for furaquinocin biosynthesis: identification of a physiological polyketide substrate and its prenylated reaction products

Furaquinocin is a natural polyketide-isoprenoid hybrid (meroterpenoid) that exhibits antitumor activity and is produced by the Streptomyces sp. strain KO-3988. Bioinformatic analysis of furaquinocin biosynthesis has identified Fur7 as a possible prenyltransferase that attaches a geranyl group to an unidentified polyketide scaffold. Here, we report the identification of a physiological polyketide substrate for Fur7, as well as its reaction product and the biochemical characterization of Fur7. A Streptomyces albus transformant (S. albus/pWHM-Fur2_del7) harboring the furaquinocin biosynthetic gene cluster lacking the fur7 gene did not produce furaquinocin but synthesized the novel intermediate 2-methoxy-3-methyl-flaviolin. After expression and purification from Escherichia coli, the recombinant Fur7 enzyme catalyzed the transfer of a geranyl group to 2-methoxy-3-methyl-flaviolin to yield 6-prenyl-2-methoxy-3-methyl-flaviolin and 7-O-geranyl-2-methoxy-3-methyl-flaviolin in a 10:1 ratio. The reaction proceeded independently of divalent cations. When 6-prenyl-2-methoxy-3-methyl-flaviolin was added to the culture medium of S. albus/pWHM-Fur2_del7, furaquinocin production was restored. The promiscuous substrate specificity of Fur7 was demonstrated with respect to prenyl acceptor substrates and prenyl donor substrates. The steady-state kinetic constants of Fur7 with each prenyl acceptor substrate were also calculated.     

Identification of products of inhibition of GES-2 beta-lactamase by tazobactam by x-ray crystallography and spectrometry

The GES-2 β-lactamase is a class A carbapenemase, the emergence of which in clinically important bacterial pathogens is a disconcerting development as the enzyme confers resistance to carbapenem antibiotics. Tazobactam is a clinically used inhibitor of class A β-lactamases, which inhibits the GES-2 enzyme effectively, restoring susceptibility to β-lactam antibiotics. We have investigated the details of the mechanism of inhibition of the GES-2 enzyme by tazobactam. By the use of UV spectrometry, mass spectroscopy, and x-ray crystallography, we have documented and identified the involvement of a total of seven distinct GES-2·tazobactam complexes and one product of the hydrolysis of tazobactam that contribute to the inhibition profile. The x-ray structures for the GES-2 enzyme are for both the native (1.45 Å) and the inhibited complex with tazobactam (1.65 Å). This is the first such structure of a carbapenemase in complex with a clinically important β-lactam inhibitor, shedding light on the structural implications for the inhibition process.     

Isolation and biological characterization of staphyloferrin B, a compound with siderophore activity from staphylococci

Abstract A highly hydrophilic compound with siderophore activity has been isolated from the supernatant of Staphylococcus hyicus DSM 20459 grown under iron-restricted conditions. The metabolite, named staphyloferrin B, is strictly iron-regulated and produced by a large variety of staphylococci strains. In vivo iron transport measurements and the growth-promoting activity in a bioassay establish staphyloferrin B as the second siderophore for staphylococci besides the previously described staphyloferrin A. The structure elucidation revealed 2,3-diaminopropionic acid, citrate, ethylenediamine and 2-ketoglutaric acid as structural components of the compound. Thus, staphyloferrin B is a structurally new siderophore of the complexone type.     

Characterization of an Actin-targeting ADP-ribosyltransferase from Aeromonas hydrophila

The mono-ADP-ribosyltransferase (mART) toxins are contributing factors to a number of human diseases, including cholera, diphtheria, traveler''s diarrhea, and whooping cough. VahC is a cytotoxic, actin-targeting mART from Aeromonas hydrophila PPD134/91. This bacterium is implicated primarily in diseases among freshwater fish species but also contributes to gastrointestinal and extraintestinal infections in humans. VahC was shown to ADP-ribosylate Arg-177 of actin, and the kinetic parameters were K_m(NAD⁺) = 6 μm, K_m(actin) = 24 μm, and k_cat = 22 s⁻¹. VahC activity caused depolymerization of actin filaments, which induced caspase-mediated apoptosis in HeLa Tet-Off cells. Alanine-scanning mutagenesis of predicted catalytic residues showed the predicted loss of in vitro mART activity and cytotoxicity. Bioinformatic and kinetic analysis also identified three residues in the active site loop that were critical for the catalytic mechanism. A 1.9 Å crystal structure supported the proposed roles of these residues and their conserved nature among toxin homologues. Several small molecules were characterized as inhibitors of in vitro VahC mART activity and suramin was the best inhibitor (IC₅₀ = 20 μm). Inhibitor activity was also characterized against two other actin-targeting mART toxins. Notably, these inhibitors represent the first report of broad spectrum inhibition of actin-targeting mART toxins.