Profiling of in vitro activities of urea-based inhibitors against cysteine synthases from Mycobacterium tuberculosis

CysK1 and CysK2 are two members of the cysteine/S-sulfocysteine synthase family in Mycobacterium tuberculosis, responsible for the de novo biosynthesis of l-cysteine, which is subsequently used as a building block for mycothiol. This metabolite is the first line defense of this pathogen against reactive oxygen and nitrogen species released by host macrophages after phagocytosis. In a previous medicinal chemistry campaign we had developed urea-based inhibitors of the cysteine synthase CysM with bactericidal activity against dormant M. tuberculosis. In this study we extended these efforts by examination of the in vitro activities of a library consisting of 71 urea compounds against CysK1 and CysK2. Binding was established by fluorescence spectroscopy and inhibition by enzyme assays. Several of the compounds inhibited these two cysteine synthases, with the most potent inhibitor displaying an IC₅₀ value of 2.5 µM for CysK1 and 6.6 µM for CysK2, respectively. Four of the identified molecules targeting CysK1 and CysK2 were also among the top ten inhibitors of CysM, suggesting that potent compounds could be developed with activity against all three enzymes.     

Assessing functional diversity in the soybean β-substituted alanine synthase enzyme family

In plants, proteins of the β-substituted alanine synthase (BSAS) enzyme family perform a diverse range of reactions, including formation of cysteine from O-acetylserine and sulfide, detoxification of cyanide by its addition to cysteine, the breakdown of cysteine into pyruvate, ammonia, and sulfide, and the synthesis of S-sulfocysteine. With the completed genome sequence of soybean (Glycine max (L.) Merr. cv. Williams 82), the functional diversity of the BSAS in this highly duplicated plant species was examined to determine whether soybean BSAS enzymes catalyze the various reactions connected to cysteine metabolism. The 16 soybean BSAS can be grouped into clades that are similar to those observed in Arabidopsis. Biochemical analysis of soybean BSAS proteins demonstrate that enzymes of clades I and III function as O-acetylserine sulfhydrylases for cysteine synthesis, clade II encodes cysteine desulfhydrase activity, and that clade V proteins function as β-cyanoalanine synthase for cyanide detoxification. Although clade IV is similar to Arabidopsis S-sulfocysteine synthase, this activity was not detected in the soybean homolog. Overall, our results show that bioinformatics approach provides a useful method to assess the biochemical properties of BSAS enzymes in plant species.     

Combatting antimicrobial resistance via the cysteine biosynthesis pathway in bacterial pathogens

Antibiotics are the cornerstone of modern medicine and agriculture, and rising antibiotic resistance is one the biggest threats to global health and food security. Identifying new and different druggable targets for the development of new antibiotics is absolutely crucial to overcome resistance. Adjuvant strategies that either enhance the activity of existing antibiotics or improve clearance by the host immune system provide another mechanism to combat antibiotic resistance. Targeting a combination of essential and non-essential enzymes that play key roles in bacterial metabolism is a promising strategy to develop new antimicrobials and adjuvants, respectively. The enzymatic synthesis of L-cysteine is one such strategy. Cysteine plays a key role in proteins and is crucial for the synthesis of many biomolecules important for defense against the host immune system. Cysteine synthesis is a two-step process, catalyzed by two enzymes. Serine acetyltransferase (CysE) catalyzes the first step to synthesize the pathway intermediate O-acetylserine, and O-acetylserine sulfhydrylase (CysK/CysM) catalyzes the second step using sulfide or thiosulfate to produce cysteine. Disruption of the cysteine biosynthesis pathway results in dysregulated sulfur metabolism, altering the redox state of the cell leading to decreased fitness, enhanced susceptibility to oxidative stress and increased sensitivity to antibiotics. In this review, we summarize the structure and mechanism of characterized CysE and CysK/CysM enzymes from a variety of bacterial pathogens, and the evidence that support targeting these enzymes for the development of new antimicrobials or antibiotic adjuvants. In addition, we explore and compare compounds identified thus far that target these enzymes.     

Substrate Specificities and Conformational Flexibility of 3-Ketosteroid 9α-Hydroxylases

KshA is the oxygenase component of 3-ketosteroid 9α-hydroxylase, a Rieske oxygenase involved in the bacterial degradation of steroids. Consistent with its role in bile acid catabolism, KshA1 from Rhodococcus rhodochrous DSM43269 had the highest apparent specificity (k_cat/K_m) for steroids with an isopropyl side chain at C17, such as 3-oxo-23,24-bisnorcholesta-1,4-diene-22-oate (1,4-BNC). By contrast, the KshA5 homolog had the highest apparent specificity for substrates with no C17 side chain (k_cat/K_m >10⁵ s⁻¹ m⁻¹ for 4-estrendione, 5α-androstandione, and testosterone). Unexpectedly, substrates such as 4-androstene-3,17-dione (ADD) and 4-BNC displayed strong substrate inhibition (K_iS ∼100 μm). By comparison, the cholesterol-degrading KshA_Mtb from Mycobacterium tuberculosis had the highest specificity for CoA-thioesterified substrates. These specificities are consistent with differences in the catabolism of cholesterol and bile acids, respectively, in actinobacteria. X-ray crystallographic structures of the KshA_Mtb·ADD, KshA1·1,4-BNC-CoA, KshA5·ADD, and KshA5·1,4-BNC-CoA complexes revealed that the enzymes have very similar steroid-binding pockets with the substrate''s C17 oriented toward the active site opening. Comparisons suggest Tyr-245 and Phe-297 are determinants of KshA1 specificity. All enzymes have a flexible 16-residue “mouth loop,” which in some structures completely occluded the substrate-binding pocket from the bulk solvent. Remarkably, the catalytic iron and α-helices harboring its ligands were displaced up to 4.4 Å in the KshA5·substrate complexes as compared with substrate-free KshA, suggesting that Rieske oxygenases may have a dynamic nature similar to cytochrome P450.     

Isoniazid Inhibits the Heme-Based Reactivity of Mycobacterium tuberculosis Truncated Hemoglobin N

Isoniazid represents a first-line anti-tuberculosis medication in prevention and treatment. This prodrug is activated by a mycobacterial catalase-peroxidase enzyme called KatG in Mycobacterium tuberculosis), thereby inhibiting the synthesis of mycolic acid, required for the mycobacterial cell wall. Moreover, isoniazid activation by KatG produces some radical species (e.g., nitrogen monoxide), that display anti-mycobacterial activity. Remarkably, the ability of mycobacteria to persist in vivo in the presence of reactive nitrogen and oxygen species implies the presence in these bacteria of (pseudo-)enzymatic detoxification systems, including truncated hemoglobins (trHbs). Here, we report that isoniazid binds reversibly to ferric and ferrous M. tuberculosis trHb type N (or group I; Mt-trHbN(III) and Mt-trHbN(II), respectively) with a simple bimolecular process, which perturbs the heme-based spectroscopic properties. Values of thermodynamic and kinetic parameters for isoniazid binding to Mt-trHbN(III) and Mt-trHbN(II) are K = (1.1±0.1)×10⁻⁴ M, k _on = (5.3±0.6)×10³ M⁻¹ s⁻¹ and k _off = (4.6±0.5)×10⁻¹ s⁻¹; and D = (1.2±0.2)×10⁻³ M, d _on = (1.3±0.4)×10³ M⁻¹ s⁻¹, and d _off = 1.5±0.4 s⁻¹, respectively, at pH 7.0 and 20.0°C. Accordingly, isoniazid inhibits competitively azide binding to Mt-trHbN(III) and Mt-trHbN(III)-catalyzed peroxynitrite isomerization. Moreover, isoniazid inhibits Mt-trHbN(II) oxygenation and carbonylation. Although the structure of the Mt-trHbN-isoniazid complex is not available, here we show by docking simulation that isoniazid binding to the heme-Fe atom indeed may take place. These data suggest a direct role of isoniazid to impair fundamental functions of mycobacteria, e.g. scavenging of reactive nitrogen and oxygen species, and metabolism.     

Kinetic Characterization and Phosphoregulation of the Francisella tularensis 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (MEP Synthase)

Deliberate and natural outbreaks of infectious disease underscore the necessity of effective vaccines and antimicrobial/antiviral therapeutics. The prevalence of antibiotic resistant strains and the ease by which antibiotic resistant bacteria can be intentionally engineered further highlights the need for continued development of novel antibiotics against new bacterial targets. Isoprenes are a class of molecules fundamentally involved in a variety of crucial biological functions. Mammalian cells utilize the mevalonic acid pathway for isoprene biosynthesis, whereas many bacteria utilize the methylerythritol phosphate (MEP) pathway, making the latter an attractive target for antibiotic development. In this report we describe the cloning and characterization of Francisella tularensis MEP synthase, a MEP pathway enzyme and potential target for antibiotic development. In vitro growth-inhibition assays using fosmidomycin, an inhibitor of MEP synthase, illustrates the effectiveness of MEP pathway inhibition with F. tularensis. To facilitate drug development, F. tularensis MEP synthase was cloned, expressed, purified, and characterized. Enzyme assays produced apparent kinetic constants (K_M^DXP = 104 µM, K_M^NADPH = 13 µM, k_cat^DXP = 2 s⁻¹, k_cat^NADPH = 1.3 s⁻¹), an IC₅₀ for fosmidomycin of 247 nM, and a K_i for fosmidomycin of 99 nM. The enzyme exhibits a preference for Mg⁺² as a divalent cation. Titanium dioxide chromatography-tandem mass spectrometry identified Ser177 as a site of phosphorylation. S177D and S177E site-directed mutants are inactive, suggesting a mechanism for post-translational control of metabolic flux through the F. tularensis MEP pathway. Overall, our study suggests that MEP synthase is an excellent target for the development of novel antibiotics against F. tularensis.     

Actinobacterial Acyl Coenzyme A Synthetases Involved in Steroid Side-Chain Catabolism

Bacterial steroid catabolism is an important component of the global carbon cycle and has applications in drug synthesis. Pathways for this catabolism involve multiple acyl coenzyme A (CoA) synthetases, which activate alkanoate substituents for β-oxidation. The functions of these synthetases are poorly understood. We enzymatically characterized four distinct acyl-CoA synthetases from the cholate catabolic pathway of Rhodococcus jostii RHA1 and the cholesterol catabolic pathway of Mycobacterium tuberculosis. Phylogenetic analysis of 70 acyl-CoA synthetases predicted to be involved in steroid metabolism revealed that the characterized synthetases each represent an orthologous class with a distinct function in steroid side-chain degradation. The synthetases were specific for the length of alkanoate substituent. FadD19 from M. tuberculosis H37Rv (FadD19_Mtb) transformed 3-oxo-4-cholesten-26-oate (k_cat/K_m = 0.33 × 10⁵ ± 0.03 × 10⁵ M⁻¹ s⁻¹) and represents orthologs that activate the C₈ side chain of cholesterol. Both CasG_RHA1 and FadD17_Mtb are steroid-24-oyl-CoA synthetases. CasG and its orthologs activate the C₅ side chain of cholate, while FadD17 and its orthologs appear to activate the C₅ side chain of one or more cholesterol metabolites. CasI_RHA1 is a steroid-22-oyl-CoA synthetase, representing orthologs that activate metabolites with a C₃ side chain, which accumulate during cholate catabolism. CasI had similar apparent specificities for substrates with intact or extensively degraded steroid nuclei, exemplified by 3-oxo-23,24-bisnorchol-4-en-22-oate and 1β(2′-propanoate)-3aα-H-4α(3″-propanoate)-7aβ-methylhexahydro-5-indanone (k_cat/K_m = 2.4 × 10⁵ ± 0.1 × 10⁵ M⁻¹ s⁻¹ and 3.2 × 10⁵ ± 0.3 × 10⁵ M⁻¹ s⁻¹, respectively). Acyl-CoA synthetase classes involved in cholate catabolism were found in both Actinobacteria and Proteobacteria. Overall, this study provides insight into the physiological roles of acyl-CoA synthetases in steroid catabolism and a phylogenetic classification enabling prediction of specific functions of related enzymes.     

Two Structurally Different Dienelactone Hydrolases (TfdEI and TfdEII) from Cupriavidus necator JMP134 Plasmid pJP4 Catalyse Cis- and Trans-Dienelactones with Similar Efficiency

In this study, dienelactone hydrolases (TfdEI and TfdEII) located on plasmid pJP4 of Cupriavidus necator JMP134 were cloned, purified, characterized and three dimensional structures were predicted. tfdEI and tfdEII genes were cloned into pET21b vector and expressed in E. coli BL21(DE3). The enzymes were purified by applying ultra-membrane filtration, anion-exchange QFF and gel-filtration columns. The enzyme activity was determined by using cis-dienelactone. The three-dimensional structure of enzymes was predicted using SWISS-MODEL workspace and the biophysical properties were determined on ExPASy server. Both TfdEI and TfdEII (M_r 25 kDa) exhibited optimum activity at 37°C and pH 7.0. The enzymes retained approximately 50% of their activity after 1 h of incubation at 50°C and showed high stability against denaturing agents. The TfdEI and TfdEII hydrolysed cis-dienelactone at a rate of 0.258 and 0.182 µMs⁻¹, with a K_m value of 87 µM and 305 µM, respectively. Also, TfdEI and TfdEII hydrolysed trans-dienelactone at a rate of 0.053 µMs⁻¹ and 0.0766 µMs⁻¹, with a K_m value of 84 µM and 178 µM, respectively. The TfdEI and TfdEII k_cat/K_m ratios were 0.12 µM⁻¹s⁻¹and 0.13 µM⁻¹s⁻¹ and 0.216 µM⁻¹s⁻¹ and 0.094 µM⁻¹s⁻¹ for for cis- and trans-dienelactone, respectively. The k_cat/K_m ratios for cis-dienelactone show that both enzymes catalyse the reaction with same efficiency even though K_m value differs significantly. This is the first report to characterize and compare reaction kinetics of purified TfdEI and TfdEII from Cupriavidus necator JMP134 and may be helpful for further exploration of their catalytic mechanisms.     

Cloning,purification, kinetic and anion inhibition studies of a recombinant β-carbonic anhydrase from the Atlantic salmon parasite platyhelminth Gyrodactylus salaris

A β-class carbonic anhydrase (CA, EC 4.2.1.1) was cloned from the genome of the Monogenean platyhelminth Gyrodactylus salaris, a parasite of Atlantic salmon. The new enzyme, GsaCAβ has a significant catalytic activity for the physiological reaction, CO₂ + H₂O ⇋ HCO₃⁻ + H⁺ with a k_cat of 1.1 × 10⁵ s⁻¹ and a k_cat/K_m of 7.58 × 10⁶ M⁻¹ × s⁻¹. This activity was inhibited by acetazolamide (K_I of 0.46 µM), a sulphonamide in clinical use, as well as by selected inorganic anions and small molecules. Most tested anions inhibited GsaCAβ at millimolar concentrations, but sulfamide (K_I of 81 µM), N,N-diethyldithiocarbamate (K_I of 67 µM) and sulphamic acid (K_I of 6.2 µM) showed a rather efficient inhibitory action. There are currently very few non-toxic agents effective in combating this parasite. GsaCAβ is subsequently proposed as a new drug target for which effective inhibitors can be designed.     

Endogenous Inhibitors for Pollen Germination and Pollen Tube Elongation in Pinus densiflora SIEB,et Zucc.

Sulfur metabolism in Cephalosporium acremonium was investigated using a mutant, 8650⁺/ OAH^?/SeMe^R, which could not convert cysteine or inorganic sulfur to methionine. The production of cephalosporin by the mutant depended on the amount of S-sulfocysteine in a chemically defined medium supplemented with a low level of methionine sufficient to support optimal growth. S-Sulfocysteine was detected in an extract of cells grown in the presence of sodium thiosulfate and l-serine. Furthermore, an NADPH-linked reduction of S-sulfocysteine to cysteine was demonstrated in a cell-free extract. These facts suggest that S-sulfocysteine is a direct precursor in cysteine biosynthesis in C. acremonium and an alternative pathway involving the compound is one of the most important ones in cephalosporin C production by this fungus.     

The C-terminal of CysM from Mycobacterium tuberculosis protects the aminoacrylate intermediate and is involved in sulfur donor selectivity

A new crystal structure of the dimeric cysteine synthase CysM from Mycobacterium tuberculosis reveals an open and a closed conformation of the enzyme. In the closed conformation the five carboxy-terminal amino acid residues are inserted into the active site cleft. Removal of this segment results in a decreased lifetime of the α-aminoacrylate reaction intermediate, an increased sensitivity to oxidants such as hydrogen peroxide, and loss of substrate selectivity with respect to the sulfur carrier thiocarboxylated CysO. These results highlight features of CysM that might be of particular importance for cysteine biosynthesis under oxidative stress in M. tuberculosis.     

Efficient one-step production of (S)-1-phenyl-1,2-ethanediol from (R)-enantiomer plus NAD+–NADPH in-situ regeneration using engineered Escherichia coli

Background

Escherichia coli has two L-cysteine biosynthetic pathways; one is synthesized from O-acetyl L-serine (OAS) and sulfate by L-cysteine synthase (CysK), and another is produced via S-sulfocysteine (SSC) from OAS and thiosulfate by SSC synthase (CysM). SSC is converted into L-cysteine and sulfite by an uncharacterized reaction. As thioredoxins (Trx1 and Trx2) and glutaredoxins (Grx1, Grx2, Grx3, Grx4, and NrdH) are known as reductases of peptidyl disulfides, overexpression of such reductases might be a good way for improving L-cysteine production to accelerate the reduction of SSC in E. coli.

Results

Because the redox enzymes can reduce the disulfide that forms on proteins, we first tested whether these enzymes catalyze the reduction of SSC to L-cysteine. All His-tagged recombinant enzymes, except for Grx4, efficiently convert SSC into L-cysteine in vitro. Overexpression of Grx1 and NrdH enhanced a 15-40% increase in the E. coliL-cysteine production. On the other hand, disruption of the cysM gene cancelled the effect caused by the overexpression of Grx1 and NrdH, suggesting that its improvement was due to the efficient reduction of SSC under the fermentative conditions. Moreover, L-cysteine production in knockout mutants of the sulfite reductase genes (ΔcysI and ΔcysJ) and the L-cysteine synthase gene (ΔcysK) each decreased to about 50% of that in the wild-type strain. Interestingly, there was no significant difference in L-cysteine production between wild-type strain and gene deletion mutant of the upstream pathway of sulfite (ΔcysC or ΔcysH). These results indicate that sulfite generated from the SSC reduction is available as the sulfur source to produce additional L-cysteine molecule. It was finally found that in the E. coliL-cysteine producer that co-overexpress glutaredoxin (NrdH), sulfite reductase (CysI), and L-cysteine synthase (CysK), there was the highest amount of L-cysteine produced per cell.

Conclusions

In this work, we showed that Grx1 and NrdH reduce SSC to L-cysteine, and the generated sulfite is then utilized as the sulfur source to produce additional L-cysteine molecule through the sulfate pathway in E. coli. We also found that co-overexpression of NrdH, CysI, and CysK increases L-cysteine production. Our results propose that the enhancement of thioredoxin/glutaredoxin-mediated L-cysteine synthesis from SSC is a novel method for improvement of L-cysteine production.     

A Flavin-dependent Monooxygenase from Mycobacterium tuberculosis Involved in Cholesterol Catabolism

Mycobacterium tuberculosis (Mtb) and Rhodococcus jostii RHA1 have similar cholesterol catabolic pathways. This pathway contributes to the pathogenicity of Mtb. The hsaAB cholesterol catabolic genes have been predicted to encode the oxygenase and reductase, respectively, of a flavin-dependent mono-oxygenase that hydroxylates 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (3-HSA) to a catechol. An hsaA deletion mutant of RHA1 did not grow on cholesterol but transformed the latter to 3-HSA and related metabolites in which each of the two keto groups was reduced: 3,9-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-17-one (3,9-DHSA) and 3,17-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9-one (3,17-DHSA). Purified 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione 4-hydroxylase (HsaAB) from Mtb had higher specificity for 3-HSA than for 3,17-DHSA (apparent k_cat/K_m = 1000 ± 100 m⁻¹ s⁻¹ versus 700 ± 100 m⁻¹ s⁻¹). However, 3,9-DHSA was a poorer substrate than 3-hydroxybiphenyl (apparent k_cat/K_m = 80 ± 40 m⁻¹ s⁻¹). In the presence of 3-HSA the K_mapp for O₂ was 100 ± 10 μm. The crystal structure of HsaA to 2.5-Å resolution revealed that the enzyme has the same fold, flavin-binding site, and catalytic residues as p-hydroxyphenyl acetate hydroxylase. However, HsaA has a much larger phenol-binding site, consistent with the enzyme''s substrate specificity. In addition, a second crystal form of HsaA revealed that a C-terminal flap (Val³⁶⁷–Val³⁹⁴) could adopt two conformations differing by a rigid body rotation of 25° around Arg³⁶⁶. This rotation appears to gate the likely flavin entrance to the active site. In docking studies with 3-HSA and flavin, the closed conformation provided a rationale for the enzyme''s substrate specificity. Overall, the structural and functional data establish the physiological role of HsaAB and provide a basis to further investigate an important class of monooxygenases as well as the bacterial catabolism of steroids.     

The Mercaptopyruvate Sulfurtransferase of Trichomonas vaginalis Links Cysteine Catabolism to the Production of Thioredoxin Persulfide

Trichomonas vaginalis is a protozoan parasite of humans that is able to synthesize cysteine de novo using cysteine synthase but does not produce glutathione. In this study, high pressure liquid chromatography analysis confirmed that cysteine is the major intracellular redox buffer by showing that T. vaginalis contains high levels of cysteine (∼600 μm) comprising more than 70% of the total thiols detected. To investigate possible mechanisms for the regulation of cysteine levels in T. vaginalis, we have characterized enzymes of the mercaptopyruvate pathway. This consists of an aspartate aminotransferase (TvAspAT1), which transaminates cysteine to form 3-mercaptopyruvate (3-MP), and mercaptopyruvate sulfurtransferase (TvMST), which transfers the sulfur of 3-MP to a nucleophilic acceptor, generating pyruvate. TvMST has high activity with 3-MP as a sulfur donor and can use several thiol compounds as sulfur acceptor substrates. Our analysis indicated that TvMST has a k_cat/K_m for reduced thioredoxin of 6.2 × 10⁷ m⁻¹ s⁻¹, more than 100-fold higher than that observed for β-mercaptoethanol and cysteine, suggesting that thioredoxin is a preferred substrate for TvMST. Thiol trapping and mass spectrometry provided direct evidence for the formation of thioredoxin persulfide as a product of this reaction. The thioredoxin persulfide could serve a biological function such as the transfer of the persulfide to a target protein or the sequestered release of sulfide for biosynthesis. Changes in MST activity of T. vaginalis in response to variation in the supply of exogenous cysteine are suggestive of a role for the mercaptopyruvate pathway in the removal of excess intracellular cysteine, redox homeostasis, and antioxidant defense.     

Nucleoside diphosphate kinase from Mycobacterium tuberculosis cleaves single strand DNA within the human c-myc promoter in an enzyme-catalyzed reaction

The reason for secretion of nucleoside diphosphate kinase (NdK), an enzyme involved in maintaining the cellular pool of nucleoside triphosphates in both prokaryotes and eukaryotes, by Mycobacterium tuberculosis is intriguing. We recently observed that NdK from M.tuberculosis (mNdK) localizes within nuclei of HeLa and COS-1 cells and also nicks chromosomal DNA in situ (A. K. Saini, K. Maithal, P. Chand, S. Chowdhury, R. Vohra, A. Goyal, G. P. Dubey, P. Chopra, R. Chandra, A. K. Tyagi, Y. Singh and V. Tandon (2004) J. Biol. Chem., 279, 50142–50149). In the current study, using a molecular beacon approach, we demonstrate that the mNdK catalyzes the cleavage of single strand DNA. It displays Michaelis–Menten kinetics with a k_cat/K_M of 9.65 (±0.88) × 10⁶ M⁻¹ s⁻¹. High affinity (K_d ≈ K_M of ~66 nM) and sequence-specific binding to the sense strand of the nuclease hypersensitive region in the c-myc promoter was observed. This is the first study demonstrating that the cleavage reaction is also enzyme-catalyzed in addition to the enzymatic kinase activity of multifunctional NdK. Using our approach, we demonstrate that GDP competitively inhibits the nuclease activity with a K_I of ~1.9 mM. Recent evidence implicates mNdK as a potent virulence factor in tuberculosis owing to its DNase-like activity. In this context, our results demonstrate a molecular mechanism that could be the basis for assessing in situ DNA damage by secretory mNdK.     

The Cysteine Desulfhydrase CdsH Is Conditionally Required for Sulfur Mobilization to the Thiamine Thiazole in Salmonella enterica

Thiamine pyrophosphate is a required coenzyme that contains a mechanistically important sulfur atom. In Salmonella enterica, sulfur is trafficked to both thiamine biosynthesis and 4-thiouridine biosynthesis by the enzyme ThiI using persulfide (R-S-S-H) chemistry. It was previously reported that a thiI mutant strain could grow independent of exogenous thiamine in the presence of cysteine, suggesting there was a second mechanism for sulfur mobilization. Data reported here show that oxidation products of cysteine rescue the growth of a thiI mutant strain by a mechanism that requires the transporter YdjN and the cysteine desulfhydrase CdsH. The data are consistent with a model in which sulfide produced by CdsH reacts with cystine (Cys-S-S-Cys), S-sulfocysteine (Cys-S-SO₃⁻), or another disulfide to form a small-molecule persulfide (R-S-S-H). We suggest that this persulfide replaced ThiI by donating sulfur to the thiamine sulfur carrier protein ThiS. This model describes a potential mechanism used for sulfur trafficking in organisms that lack ThiI but are capable of thiamine biosynthesis.     

Characterization of a Novel Metal-Dependent D-Psicose 3-Epimerase from Clostridium scindens 35704

The noncharacterized protein CLOSCI_02528 from Clostridium scindens ATCC 35704 was characterized as D-psicose 3-epimerase. The enzyme showed maximum activity at pH 7.5 and 60°C. The half-life of the enzyme at 50°C was 108 min, suggesting the enzyme was relatively thermostable. It was strictly metal-dependent and required Mn²⁺ as optimum cofactor for activity. In addition, Mn²⁺ improved the structural stability during both heat- and urea-induced unfolding. Using circular dichroism measurements, the apparent melting temperature (T _m) and the urea midtransition concentration (C _m) of metal-free enzyme were 64.4°C and 2.68 M. By comparison, the Mn²⁺-bound enzyme showed higher T _m and C _m with 67.3°C and 5.09 M. The Michaelis-Menten constant (K _m), turnover number (k _cat), and catalytic efficiency (k _cat/K _m) values for substrate D-psicose were estimated to be 28.3 mM, 1826.8 s⁻¹, and 64.5 mM⁻¹ s⁻¹, respectively. The enzyme could effectively produce D-psicose from D-fructose with the turnover ratio of 28%.     

Identification of a 5′-Deoxyadenosine Deaminase in Methanocaldococcus jannaschii and Its Possible Role in Recycling the Radical S-Adenosylmethionine Enzyme Reaction Product 5′-Deoxyadenosine

We characterize here the MJ1541 gene product from Methanocaldococcus jannaschii, an enzyme that was annotated as a 5′-methylthioadenosine/S-adenosylhomocysteine deaminase (EC 3.5.4.31/3.5.4.28). The MJ1541 gene product catalyzes the conversion of 5′-deoxyadenosine to 5′-deoxyinosine as its major product but will also deaminate 5′-methylthioadenosine, S-adenosylhomocysteine, and adenosine to a small extent. On the basis of these findings, we are naming this new enzyme 5′-deoxyadenosine deaminase (DadD). The K_m for 5′-deoxyadenosine was found to be 14.0 ± 1.2 μM with a k_cat/K_m of 9.1 × 10⁹ M⁻¹ s⁻¹. Radical S-adenosylmethionine (SAM) enzymes account for nearly 2% of the M. jannaschii genome, where the major SAM derived products is 5′-deoxyadenosine. Since 5′-dA has been demonstrated to be an inhibitor of radical SAM enzymes; a pathway for removing this product must be present. We propose here that DadD is involved in the recycling of 5′-deoxyadenosine, whereupon the 5′-deoxyribose moiety of 5′-deoxyinosine is further metabolized to deoxyhexoses used for the biosynthesis of aromatic amino acids in methanogens.     

Search for polythionates in cultures of Chromatium vinosum after sulfide incubation

Cultures of Chromatium vinosum, devoid of sulfur globules, were supplemented with sulfide and incubated under anoxic conditions in the light. The concentrations of sulfide, polysulfides, thiosulfate, polythionates and elemental sulfur (sulfur rings) were monitored for 3 days by ion-chromatography and reversed-phase HPLC. While sulfide disappeared rapidly, thiosulfate and elemental sulfur (S₆, S₇ S₈ rings) were formed. After sulfide depletion, the concentration of thiosulfate decreased fairly rapidly, but elemental sulfur was oxidized very slowly to sulfate. Neither polysulfides (S _x ^2– ), polythionates (S_nO ₆ ^2– , n=4–6), nor other polysulfur compounds could be detected, which is in accordance with the fact that sulfide-grown cells were able to oxidize polysulfide without lag. The nature of the intracellular sulfur globules is discussed.