Search

Purine bases and nucleosides are produced by turnover of nucleotides and nucleic acids as well as from some cellular metabolic pathways. Adenosine released from the S -adenosyl-L-methionine cycle is linked to many methyltransferase reactions, such as the biosynthesis of caffeine and glycine betaine. Adenine is produced by the methionine cycles, which is related to other biosynthesis pathways, such those for the production of ethylene, nicotianamine and polyamines. These purine compounds are recycled for nucleotide biosynthesis by so-called “salvage pathways”. However, the salvage pathways are not merely supplementary routes for nucleotide biosynthesis, but have essential functions in many plant processes. In plants, the major salvage enzymes are adenine phosphoribosyltransferase (EC 2.4.2.7) and adenosine kinase (EC 2.7.1.20). AMP produced by these enzymes is converted to ATP and utilised as an energy source as well as for nucleic acid synthesis. Hypoxanthine, guanine, inosine and guanosine are salvaged to IMP and GMP by hypoxanthine/guanine phosphoribosyltransferase (EC 2.4.2.8) and inosine/guanosine kinase (EC 2.7.1.73). In contrast to de novo purine nucleotide biosynthesis, synthesis by the salvage pathways is extremely favourable, energetically, for cells. In addition, operation of the salvage pathway reduces the intracellular levels of purine bases and nucleosides which inhibit other metabolic reactions. The purine salvage enzymes also catalyse the respective formation of cytokinin ribotides, from cytokinin bases, and cytokinin ribosides. Since cytokinin bases are the active form of cytokinin hormones, these enzymes act to maintain homeostasis of cellular cytokinin bioactivity. This article summarises current knowledge of purine salvage pathways and their possible function in plants and purine salvage activities associated with various physiological phenomena are reviewed. [ABSTRACT FROM AUTHOR]

Tyrosyl-DNA phosphodiesterase (Tdp1) is a DNA 3'-end processing enzyme that repairs topoisomerase 1B-induced DNA damage. We use a new tool combining site-specific DNA-protein cross-linking with mass spectrometry to identify Tdp1 interactions with DNA. A conserved phenylalanine (F259) of Tdp1, required for efficient DNA processing in biochemical assays, cross-links to defined positions in DNA substrates. Crystal structures of Tdp1-DNA complexes capture the DNA repair machinery after 3'-end cleavage; these reveal how Tdp1 coordinates the 3'-phosphorylated product of nucleosidase activity and accommodates duplex DNA. A hydrophobic wedge splits the DNA ends, directing the scissile strand through a channel towards the active site. The F259 side-chain stacks against the -3 base pair, delimiting the junction of duplexed and melted DNA, and fixes the scissile strand in the channel. Our results explain why Tdp1 cleavage is non-processive and provide a molecular basis for DNA 3'-end processing by Tdp1. [ABSTRACT FROM AUTHOR]

S -adenosylhomocysteine/Methylthioadenosine nucleosidase (SAHN E.C.3.2.2.9) does not exist in mammalian cells but is essential for methyl recycling in numerous bacterial and protozoan species. Inhibition of this enzyme could limit synthesis of autoinducers of bacterial quorum sensing (QS), and hence, causes reduction in biofilm formation and may attenuate virulence. In this study, sahn deletion mutant of E. coli MG1655, sahn -complemented strain, and SANH-overexpressing strain were established and used to identify the secretion of autoinducer-2 (AI-2) and biofilm formation. The results indicated that deletion of the sahn gene abolished the production of the QS signal AI-2 and biofilm formation in mutant strain MG1655-Δ sahn . And the complementation strain MG1655-Δ sahn (pET-28a- sahn ) showed restored production of AI-2 and biofilm formation, which indicates that the sahn gene plays an important role in bacterial quorum sensing. The recombinant SAHN protein was overexpressed and purified. The enzymatic activity of SAHN was successfully determined by a coupling-enzyme analysis based on xanthine oxidase, with the V max and K m of SAHN enzymatic reaction confirmed. Given that sahn is essential for the quorum sensing of both Gram-negative and Gram-positive bacteria, SAHN could be a potential target for wide-spectrum antibiotics. [ABSTRACT FROM AUTHOR]

MTAN (5'-methylthioadenosine nucleosidase) catalyzes the hydrolysis of the N-ribosidic bond of a variety of adenosine-containing metabolites. The Helicobacter pylori MTAN (HpMTAN) hydrolyzes 6-amino-6-deoxyfutalosine in the second step of the alternative menaquinone biosynthetic pathway. Substrate binding of the adenine moiety is mediated almost exclusively by hydrogen bonds, and the proposed catalytic mechanism requires multiple protontransfer events. Of particular interest is the protonation state of residue D198, which possesses a pKa above 8 and functions as a general acid to initiate the enzymatic reaction. In this study we present three corefined neutron/X-ray crystal structures of wildtype HpMTAN cocrystallized with S-adenosylhomocysteine (SAH), Formycin A (FMA), and (3R,4S)-4-(4-Chlorophenylthiomethyl)-1-[(9-deazaadenin- 9-yl)methyl]-3-hydroxypyrrolidine (p-ClPh-Thio-DADMe-ImmA) as well as one neutron/X-ray crystal structure of an inactive variant (HpMTAN-D198N) cocrystallized with SAH. These results support a mechanism of D198 pKa elevation through the unexpected sharing of a proton with atom N7 of the adenine moiety possessing unconventional hydrogen-bond geometry. Additionally, the neutron structures also highlight active site features that promote the stabilization of the transition state and slight variations in these interactions that result in 100-fold difference in binding affinities between the DADMe-ImmA and ImmA analogs. [ABSTRACT FROM AUTHOR]

HopAF1 is a type III effector protein of unknown function encoded in the genomes of several strains of Pseudomonas syringae and other plant pathogens. Structural modeling predicted that HopAF1 is closely related to deamidase proteins. Deamidation is the irreversible substitution of an amide group with a carboxylate group. Several bacterial virulence factors are deamidases that manipulate the activity of specific host protein substrates. We identified Arabidopsis methylthioadenosine nucleosidase proteins MTN1 and MTN2 as putative targets of HopAF1 deamidation. MTNs are enzymes in the Yang cycle, which is essential for the high levels of ethylene biosynthesis in Arabidopsis We hypothesized that HopAF1 inhibits the host defense response by manipulating MTN activity and consequently ethylene levels. We determined that bacterially delivered HopAF1 inhibits ethylene biosynthesis induced by pathogen-associated molecular patterns and that Arabidopsis mtn1 mtn2 mutant plants phenocopy the effect of HopAF1. Furthermore, we identified two conserved asparagines in MTN1 and MTN2 from Arabidopsis that confer loss of function phenotypes when deamidated via site-specific mutation. These residues are potential targets of HopAF1 deamidation. HopAF1-mediated manipulation of Yang cycle MTN proteins is likely an evolutionarily conserved mechanism whereby HopAF1 orthologs from multiple plant pathogens contribute to disease in a large variety of plant hosts. [ABSTRACT FROM AUTHOR]

The protein 5′-methylthioadenosine/ S-adenosylhomocysteine nucleosidase (MTAN) is involved in the quorum sensing of several bacterial species, including Helicobacter pylori. In particular, these bacteria depend on MTAN for synthesis of vitamin K homologs. The residue D198 in the active site of MTAN seems to be of crucial importance, by acting as a hydrogen-bond acceptor for the ligand. In this study, we investigated the conformation and dynamics of apo and holo H. pylori MTAN ( HpMTAN), and assessed the effect of protonation of D198 by use of molecular dynamics simulations. Our results show that protonation of the active site of HpMTAN can cause a conformational transition from a closed state to an open state even in the absence of substrate, via inter-chain mechanical coupling. [ABSTRACT FROM AUTHOR]

Helicobacter pylori is a Gram-negative bacterium that colonizes the gut of over 50% of the world's population. It is responsible for most peptic ulcers and is an important risk factor for gastric cancer. Antibiotic treatment for H. pylori infections is challenging as drug resistance has developed to antibiotics with traditional mechanisms of action. H. pylori uses an unusual pathway for menaquinone biosynthesis with 5'- methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) catalyzing an essential step. We validated MTAN as a target with a transition-state analogue of the enzyme [Wang, S.; Haapalainen, A M.; Yan, F.; et al. Biochemistry 2012, 51, 6892--6894]. MTAN inhibitors will only be useful drug candidates if they can both include tight binding to the MTAN target and have the ability to penetrate the complex cell membrane found in Gram-negative H. pylori. Here we explore structural scaffolds for MTAN inhibition and for growth inhibition of cultured H. pylori. Sixteen analogues reported here are transition-state analogues of H. pylori MTAN with dissociation constants of 50 pM or below. Ten of these prevent growth of the H. pylori with IC90 values below 0.01 μg/mL . These remarkable compounds meet the criteria for potent inhibition and cell penetration. As a consequence, 10 new H. pylori antibiotic candidates are identified, all of which prevent H. pylori growth at concentrations 16--2000-fold lower than the five antibiotics, amoxicillin, metronidazole, levofloxacin, tetracyclin, and clarithromycin, commonly used to treat H. pylori infections. X-ray crystal structures of MTAN cocrystallized with several inhibitors show them to bind in the active site making interactions consistent with transition-state analogues. [ABSTRACT FROM AUTHOR]

Calcineurin B-like (CBL) proteins constitute a unique family of calcium sensor relays in plants. It is well known that CBLs detect the calcium signals elicited by a variety of abiotic stresses and relay the information to a group of serine/threonine protein kinases called CBL-interacting protein kinases (CIPKs). In this study, we found that a few CBL members can also target another group of enzymes 5′-methylthioadenosine nucleosidases (MTANs), which are encoded by two genes in Arabidopsis, AtMTAN1 and AtMTAN2 . In the yeast two-hybrid system, AtMTAN1 interacted with multiple CBL members such as CBL2, CBL3 and CBL6, whereas AtMTAN2 associated exclusively with CBL3. We further demonstrated that the CBL3-AtMTAN2 association occurs in a calcium-dependent manner, which results in a significant decrease in the enzyme activity of the AtMTAN2 protein. Taken together, these results clearly indicate that the CBL family can target at least two distinct groups of enzymes (CIPKs and MTANs), conferring an additional level of complexity on the CBL-mediated signaling networks. In addition, our finding also provides a novel molecular mechanism by which calcium signals are transduced to alter metabolite profiles in plants. [ABSTRACT FROM AUTHOR]

In Vibrio cholerae , the genes required for biofilm development are repressed by quorum sensing at high cell density due to the accumulation in the medium of two signaling molecules, cholera autoinducer 1 (CAI-1) and autoinducer 2 (AI-2). A significant fraction of toxigenic V. cholerae isolates, however, exhibit dysfunctional quorum sensing pathways. It was reported that transition state analogs of the enzyme methylthioadenosine/S-adenosylhomocysteine nucleosidase (MtnN) required to make AI-2 inhibited biofilm formation in the prototype quorum sensing-deficient strain N16961. This finding prompted us to examine the role of both autoinducers and MtnN in biofilm development and virulence gene expression in a quorum sensing-deficient genetic background. Here we show that deletion of mtnN encoding methylthioadenosine/S-adenosylhomocysteine nucleosidase, cqsA (CAI-1), and/or luxS (AI-2) do not prevent biofilm development. However, two independent mtnN mutants exhibited diminished growth rate and motility in swarm agar plates suggesting that, under certain conditions, MtnN could influence biofilm formation indirectly. Nevertheless, MtnN is not required for the development of a mature biofilm. [ABSTRACT FROM AUTHOR]

5'-Methylthioadenosine/S-adenosyl-l-homocysteine nucleosidases (MTANs) catalyze the hydrolysis of 5'-methylthioadenosine to adenine and 5-methylthioribose. The amino acid sequences of the MTANs from Vibrio cholerae (VcMTAN) and Escherichia coli (EcMTAN) are 60% identical and 75% similar. Protein structure folds and kinetic properties are similar. However, binding of transition-state analogues is dominated by favorable entropy in VcMTAN and by enthalpy in EcMTAN. Catalytic sites of VcMTAN and EcMTAN in contact with reactants differ by two residues; Ala113 and Val153 in VcMTAN are Pro113 and Ile152, respectively, in EcMTAN. We mutated the VcMTAN catalytic site residues to match those of EcMTAN in anticipation of altering its properties toward EcMTAN. Inhibition of VcMTAN by transition-state analogues required filling both active sites of the homodimer. However, in the Val153Ile mutant or double mutants, transition-state analogue binding at one site caused complete inhibition. Therefore, a single amino acid, Val153, alters the catalytic site cooperativity in VcMTAN. The transition-state analogue affinity and thermodynamics in mutant VcMTAN became even more unlike those of EcMTAN, the opposite of expectations from catalytic site similarity; thus, catalytic site contacts in VcMTAN are unable to recapitulate the properties of EcMTAN. X-ray crystal structures of EcMTAN, VcMTAN, and a multiple-site mutant of VcMTAN most closely resembling EcMTAN in catalytic site contacts show no major protein conformational differences. The overall protein architectures of these closely related proteins are implicated in contributing to the catalytic site differences. [ABSTRACT FROM AUTHOR]

Prokaryotic 5′-methylthioadenosine/ S-adenosylhomocysteine nucleosidase (MtaN) is a multifunctional enzyme that can hydrolyze S-adenosyl-L-homocysteine (SAH) and S-methyl-5′-thioadenosine (MTA) to give S-ribosyl-L-homocysteine (SRH) and S-methyl-5′-thioribose (MTR), respectively. This reaction plays a key role in several metabolic pathways, including biological methylation, polyamine biosynthesis, methionine recycling and bacterial quorum sensing. Structurally, MtaN belongs to the MtnN subfamily of the purine nucleoside phosphorylase (PNP)/uridine phosphorylase (UDP) phosphorylase family. Aeromonas hydrophila has two MtnN subfamily proteins: MtaN-1, a periplasmic protein with an N-terminal signal sequence, and MtaN-2, a cytosolic protein. In this study, MtaN-1 from Aeromonas hydrophila was successfully expressed and purified using Ni-NTA affinity, Q anion-exchange and gel-filtration chromatography. Crystals of the protein in complex with the substrate SAH were obtained and diffracted to a resolution of 1.4 Å. The crystals belonged to the trigonal space group P3121 or P3221, with unit-cell parameters a = b = 102.7, c = 118.8 Å. The asymmetric unit contained two molecules of MtaN-1 complexed with SAH. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is a major biofilm-forming pathogen, and biofilm formation remains an obstacle in the treatment of clinical S. aureus infection. Methylthioadenosine/S-adenosylhomocysteine nucleosidase (Pfs) has been implicated in methylation reactions, polyamine synthesis, vitamin synthesis, and quorum-sensing pathways. In this study, we observed that the deletion of pfs gene in S. aureus NCTC8325 reduced bacterial clumping ability and resulted in the decreased biofilm formation under both static and dynamic flow conditions in an autoinducer-2-independent manner. While the PIA amount was not affected, the pfs mutation significantly decreased the amount of eDNA present in the biofilm and the cell autolysis. Consistent with reduced autolysis, the transcription levels of the autolysin genes, lytM and atlE, were reduced in the absence of Pfs. These data suggest that Pfs promotes autolysis-dependent release of eDNA and biofilm formation in S. aureus, and our findings indicate that Pfs is a potential novel target for anti-biofilm therapy. [ABSTRACT FROM AUTHOR]

Abstract: Background: Certain drugs such as glucocorticoids may interfere with the modulation of periodontal disease. In contrast, corticosteroid treatment has been associated with a protective effect with regard to periodontal breakdown, depending on the dose, pathway, and exposure time. Considering the potential relevance of nucleotidases in coordinating the cardiovascular system and inflammation processes, the aim of this study was to investigate the nucleotidase activities in the blood serum of rats with periodontal disease exposed chronically to inhaled corticosteroids. Methods: Adult male Wistar rats (n =26) were randomly assigned to one of the following four study groups: a control group that received no intervention; a periodontal disease group that received saline solution; a ‘low dose’ group that received 30μg of budesonide daily; and a corresponding ‘high dose’ group that received 100μg daily over a 15-day time course. The hydrolysis of ATP, ADP, and AMP were analysed in blood serum. Results: Periodontal disease diminished the hydrolysis of ATP and enhanced the hydrolysis of ADP. Repeated administration of either a low or high dose in the periodontal disease model of inhaled corticosteroids reversed the observed increase in ADP hydrolysis, and only the repeated administration of low doses of inhaled corticosteroids was able to reverse the decrease in the hydrolysis of ATP induced by periodontal disease. Conclusion: The variables investigated in this study may be involved in the pathophysiology of periodontal disease and may participate in the mechanisms that mediate the development of some of the side effects of inhaled corticosteroids. [Copyright &y& Elsevier]

Highlights: [•] The first structure of B. melitensis MTAN in complex with adenine was determined. [•] Enzymatic kinetic parameters of BmMTAN were revealed. [•] The catalytic mechanism and substrate preference mode of BmMTAN were elucidated. [Copyright &y& Elsevier]

5- Methylthioadenosine/S-adenosylhomocysteine nucleosidases (MTANs) are bacterial enzymes that catalyze hydrolysis of the N-ribosidic bonds of S-methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH) to form adenine and 5-thioribosyl groups. MTANs are involved in AT-i and AI-2 bacterial quorum sensing and the unusual futalosine-based menaquinone synthetic pathway in Strepto- myces Helicobacter and Campylobacter species. Crystal structures show MTANs to be homodimers with two catalytk sites near the dimer interface. Here, we explore the cooperative ligand interactions in the homodimer of Staphylococcus aureus MTAN (SaMTAN). Kinetic analysis indicated negative catalytic cooperativity. Titration of SOMTAN with the transition-state analogue MT-DADMe-lmmA gave unequal catalytic site binding, consistent with negative binding cooperativity. Thermodynamics of MT-DADMe-lmmA binding also gave negative cooperativity, where the first site had different enthalpic and entropic properties than the second site. Cysteine reactivity in a single-cysteine catalytic site loop construct of SaMTAN is reactive in native enzyme, less reactive when inhibitor is bound to one subunit, and nonreactive upon saturation with inhibitor. A fusion peptide heterodimer construct with one inactive subunit (E173Q) and one native subunit gave 25% of native SaMTAN activity, similar to native SaMTAN with MT-DADMe-lnsmA at one catalytic site. Pre.steady-state kinetics showed fast chemistry at one catalytic site, consistent with slow adenine release before catalysis occurs at the second catalytic site. The results support the two catalytic sites acting sequentially, with negative cooperativity and product release being linked to motion of a catalytic site loop contributed by the neighboring subunit. [ABSTRACT FROM AUTHOR]

Autoinducer-2 (AI-2) molecules are one class of signalling molecules involved in gene regulation dependent on population density in a mechanism commonly referred to as quorum sensing (QS). AI-2 is produced by the methylthioadenosine/S-adenosyl-homocysteine nucleosidase LuxS. In the present study, we characterise the function of bifidobacterial LuxS proteins to address the question whether these economically important bacteria are able to perform QS communication. All publically available genome sequences of bifidobacteria harbour putative luxS genes. The deduced amino acid sequences are well conserved in the genus and show good homology to the LuxS protein of the prototypical AI-2 producer Vibrio harveyi. The luxS genes of three bifidobacterial strains were successfully expressed in AI-2-negative Escherichia coli DH5α. Supernatants of these recombinant E. coli strains contained significant AI-2 activity. In initial experiments, we failed to detect AI-2 activity in supernatants of bifidobacteria grown in MRSc. High concentration of glucose as well as acidic pH had strong inhibitory effects on AI-2 activity. AI-2 activity could be detected when lower volumes of supernatants were used in the assay. Homologous overexpression of luxS in Bifidobacterium longum NCC2705 increased AI-2 levels in the supernatant. Furthermore, over-expression of luxS or supplementation with AI-2-containing supernatants enhanced biofilm formation of B. longum NCC2705. Collectively, these results suggest that bifidobacteria indeed harbour functional luxS genes that are involved in the production of AI-2-like molecules. To the best of our knowledge, this represents the first report on AI-2 activity produced by bifidobacteria. Self-produced AI-2 activity as well as AI-2-like molecules of other bacteria of the intestinal tract may have a regulatory function in biofilm formation and host colonization by bifidobacteria. [ABSTRACT FROM AUTHOR]

The recently discovered futalosine pathway is a promising target for the development of new antibiotics. The enzymes involved in this pathway are crucial for the biosynthesis of the essential prokaryotic respiratory compound menaquinone, and as the pathway is limited to few bacterial species such as the gastric pathogen Helicobacter pylori it is a potential target for specific antibiotics. In this report, the crystal structure of an H. pylori methylthioadenosine nucleosidase (MTAN; an enzyme with broad specificity and activity towards 6-amino- 6-deoxyfutalosine), which is involved in the second step of menaquinone biosynthesis, has been elucidated at a resolution of 1.76 Å and refined with R factors of Rwork = 17% and Rfree = 21%. Activity studies on the wild type and active-site mutants show that the hydrolysis of 6-amino-6-deoxyfutalosine follows a mechanism similar to that of Escherichia coli MTAN. Further evidence for this mode of action is supplied by the crystal structures of active-site mutants. Through the use of reaction intermediates, the structures give additional evidence for the previously proposed nucleosidase mechanism. These structures and the confirmed reaction mechanism will provide a structural basis for the design of new inhibitors targeting the futalosine pathway. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis is a chronicand lethal parasite disease against which no human vaccine is available.Hepato- splenomegaly and a progressive suppression of the cellular immune response are among its most important clinical signs. The characteristic cellular immunosupression was described as being mediated in part, through the spatial segregation of dendritic cells (DCs) and T cell lymphocytes due to altered frequencies and migration capabilities of DCs. In this investigation, we measured the spleen/body relative weight, the spleen parasite load and the total counts of spleen DCs of C57BL6 mice infected with Leishmania chagasi. All the variables achieved their maximum at 30 days after infection. We detected in infected animals a 5.08 fold increase of spleen relative weight, a 19.6 fold increase of parasite load and a 4.55 increase of total DCs counts, when compared to naïve controls. We further analysed the efficacy of the NH36 and F3 vaccines formulated in saponin in prevention of visceral leishmaniasis. When compared to the infected controls, both vaccines determined strong protection. The F3 vaccine induced the highest efficacy showing 95% and 49% reduction the parasite load and splenomegaly, respectively. The NH36 vaccine, on the other hand, developed a slightly lower but still significant protection reducing by 87% the parasite load and by 39% the spleen relative weight. Both vaccines also prevented the increase in total counts of DCs with no significant difference between them (36% by the NH36 and 26% by the F3 vaccine). Our results suggest that vaccination against murine visceral leishmaniasis with the NH36 vaccine can prevent the development of the disease by preventing the DCs dysfunction-related immunosupression. Additionally, they disclose the potential use of the NH36 C-terminal moiety, the F3 peptide for optimization of the vaccine efficacy. [ABSTRACT FROM AUTHOR]

Transition-state analogues of bacterial5′-methylthioadenosine/S-adenosylhomocysteinenucleosidases (MTANs) disrupt quorum-sensingpathways in Escherichia coliand Vibrio cholerae, demonstrating the potential to limit pathogenicity without placingbacteria under intense selective pressure that leads to antibioticresistance. Despite the similarity of the crystal structures of E. coliMTAN (EcMTAN) and V. choleraeMTAN (VcMTAN) boundto DADMe-Immucillin-A transition-state (TS) analogues, EcMTAN demonstrates femtomolar affinity for BuT-DADMe-Immucillin-A(BDIA) whereas VcMTAN possesses only picomolar affinity.Protein dynamic interactions are therefore implicated in this inhibitoraffinity difference. We conducted molecular dynamics simulations ofboth EcMTAN and VcMTAN in complexwith BDIA to explore differences in protein dynamic architecture.Simulations revealed that electrostatic and hydrophobic interactionswith BDIA are similar for both enzymes and thus unlikely to accountfor the difference in inhibitor affinity. The EcMTAN–BDIAcomplex reveals a greater flexibility and conformational freedom ofcatalytically important atoms. We propose that conserved motions relatedto the EcMTAN transition state correlate with theincreased affinity of BDIA for EcMTAN. Transition-stateanalogues permitting protein motion related to formation of the transitionstate are better mimics of the enzymatic transition state and canbind more tightly than those immobilizing catalytic site dynamics. [ABSTRACT FROM AUTHOR]

Sleeping sickness is a deadly disease that primarily affects sub-Saharan Africa and is caused by protozoan parasites of the Trypanosoma genus. Trypanosomes are purine auxotrophs and their uptake pathway has long been appreciated as an attractive target for drug design. Recently, one tight-binding competitive inhibitor of the trypanosomal purine-specific nucleoside hydrolase (IAGNH) showed remarkable trypanocidal activity in a murine model of infection. Here, the enzymatic characterization of T. brucei brucei IAGNH is presented, together with its high-resolution structures in the unliganded form and in complexes with different inhibitors, including the trypanocidal compound UAMC-00363. A description of the crucial contacts that account for the high-affinity inhibition of IAGNH by iminoribitol-based compounds is provided and the molecular mechanism underlying the conformational change necessary for enzymatic catalysis is identified. It is demonstrated for the first time that metalorganic complexes can compete for binding at the active site of nucleoside hydrolase enzymes, mimicking the positively charged transition state of the enzymatic reaction. Moreover, we show that divalent metal ions can act as noncompetitive IAGNH inhibitors, stabilizing a nonproductive conformation of the catalytic loop. These results open a path for rational improvement of the potency and the selectivity of existing compounds and suggest new scaffolds that may be used as blueprints for the design of novel antitrypanosomal compounds. [ABSTRACT FROM AUTHOR]

Abstract: Staphylococcus aureus is a major cause of infectious morbidity and mortality in both community and hospital settings. The bacterium continues to cause diverse invasive, life-threatening infections, such as pneumonia, endocarditis, and septicemia. Methylthioadenosine/S-adenosylhomocysteine nucleosidase (Pfs) is predicted to be an important enzyme involved in methylation reactions, polyamine synthesis, vitamin synthesis, and quorum sensing pathways. For the first time, we demonstrate that Pfs is essential for the virulence of S. aureus. The pfs mutant strain, as compared to the isogenic wild type, displayed a decreased production of extracellular proteases, which was correlated with a dramatic decrease in the expression of the sspABC operon and a moderate decrease of aur expression. The mouse model of sepsis and subcutaneous abscesses indicated that the pfs mutant strain displayed highly impaired virulence compared to the isogenic wild type. The decreased virulence of the pfs mutant strain is in correspondence with its decreased proliferation in vivo, indicated with a real-time analysis in the transparent system of zebrafish embryos. These phenotypes of the pfs mutant strain are LuxS/AI-2 independent despite the essential role pfs plays in AI-2 production. Our data suggest that Pfs is a potential novel target for anti-infection therapy. [Copyright &y& Elsevier]

The bacterial 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) enzyme is a multifunctional enzyme that catalyzes the hydrolysis of the N-ribosidic bond of at least four different adenosine-based metabolites: S-adenosylhomocysteine (SAH), 5'-methylthioadenosine (MTA), 5'-deoxyadenosine (5'-DOA), and 6-amino-6-deoxyfutalosine. These activities place the enzyme at the hub of seven fundamental bacterial metabolic pathways: S-adenosylmethionine (SAM) utilization, polyamine biosynthesis, the purine salvage pathway, the methionine salvage pathway, the SAM radical pathways, autoinducer-2 biosynthesis, and menaquinone biosynthesis. The last pathway makes MTAN essential for Helicobacter pylori viability. Although structures of various bacterial and plant MTANs have been described, the interactions between the homocysteine moiety of SAH and the 5'-alkylthiol binding site of MTAN have never been resolved. We have determined crystal structures of an inactive mutant form of H. pylori MTAN bound to MTA and SAH to 1.63 and 1.20 Å, respectively. The active form of MTAN was also crystallized in the presence of SAH, allowing the determination of the structure of a ternary enzyme-product complex resolved at 1.50 Å. These structures identify interactions between the homocysteine moiety and the 5'-alkylthiol binding site of the enzyme. This information can be leveraged for the development of species-specific MTAN inhibitors that prevent the growth of H. pylori. [ABSTRACT FROM AUTHOR]

Abstract: Several acyclic hydroxy-methylthio-amines with 3–5 carbon atoms were prepared and coupled via a methylene link to 9-deazaadenine. The products were tested for inhibition against human MTAP and Escherichia coli and Neisseria meningitidis MTANs and gave K i values as low as 0.23nM. These results were compared to those obtained with 1st and 2nd generation inhibitors (1S)-1-(9-deazaadenin-9-yl)-1,4-dideoxy-1,4-imino-5-methylthio-d-ribitol (MT-Immucillin-A, 3) and (3R,4S)-1-[9-deazaadenin-9-yl)methyl]3-hydroxy-4-methylthiomethylpyrrolidine (MT-DADMe-Immucillin-A, 4). The best inhibitors were found to exhibit binding affinities of approximately 2- to 4-fold those of 3 but were significantly weaker than 4. Cleavage of the 2,3 carbon–carbon bond in MT-Immucillin-A (3) gave an acyclic product (79) with a 21,500 fold loss of activity against E. coli MTAN. In another case, N-methylation of a side chain secondary amine resulted in a 250-fold loss of activity against the same enzyme [(±)-65 vs (±)-68]. The inhibition results were also contrasted with those acyclic derivatives previously prepared as inhibitors for a related enzyme, purine nucleoside phosphorylase (PNP), where some inhibitors in the latter case were found to be more potent than their cyclic counterparts. [Copyright &y& Elsevier]

Summary [ABSTRACT FROM AUTHOR]

Nucleosome deposition occurs on newly synthesized DNA during DNA replication and on transcriptionally active genes via nucleosome-remodeling complexes recruited by activator proteins and elongating RNA polymerase II. It has been long believed that histone deposition involves stable H3-H4 tetramers, such that newly deposited nucleosomes do not contain H3 and H4 molecules with their associated histone modifications from preexisting nucleosomes. However, biochemical analyses and recent experiments in mammalian cells have raised the idea that preexisting H3-H4 tetramers might split into dimers, resulting in mixed nucleosomes composed of "old" and "new" histones. It is unknown to what extent different genomic loci might utilize such a mechanism and under which circumstances. Here, we address whether tetramer splitting occurs in a locus-specific manner by using sequential chromatin immunoprecipitation of mononucleosomes from yeast cells containing two differentially tagged versions of H3 that are expressed "old" and "new" histones. At many genomic loci, we observe little or no nucleosomal cooccupancy of old and new H3, indicating that tetramer splitting is generally infrequent. However, cooccupancy is detected at highly active genes, which have a high rate of histone exchange. Thus, DNA replication largely results in nucleosomes bearing exclusively old or new H3-H4, thereby precluding the acquisition of new histone modifications based on preexisting modifications within the same nucleosome. In contrast, tetramer splitting, dimer exchange, and nucleosomes with mixed H3-H4 tetramers occur at highly active genes, presumably linked to rapid histone exchange associated with robust transcription. [ABSTRACT FROM AUTHOR]

Abstract: 5′-Methylthioadenosine/S-adenosylhomocysteine (MTA/SAH) nucleosidase (MTAN) plays a key role in the methionine-recycling pathway of bacteria and plants. Despite extensive structural and biochemical studies, the molecular mechanism of substrate specificity for MTAN remains an outstanding question. Bacterial MTANs show comparable efficiency in hydrolyzing MTA and SAH, while the plant enzymes select preferentially for MTA, with either no or significantly reduced activity towards SAH. Bacterial and plant MTANs show significant conservation in the overall structure, and the adenine- and ribose-binding sites. The observation of a more constricted 5′-alkylthio binding site in Arabidopsis thaliana AtMTAN1 and AtMTAN2, two plant MTAN homologues, led to the hypothesis that steric hindrance may play a role in substrate selection in plant MTANs. We show using isothermal titration calorimetry that SAH binds to both Escherichia coli MTAN (EcMTAN) and AtMTAN1 with comparable micromolar affinity. To understand why AtMTAN1 can bind but not hydrolyze SAH, we determined the structure of the protein–SAH complex at 2.2Å resolution. The lack of catalytic activity appears to be related to the enzyme’s inability to bind the substrate in a catalytically competent manner. The role of dynamics in substrate selection was also examined by probing the amide proton exchange rates of EcMTAN and AtMTAN1 via deuterium–hydrogen exchange coupled mass spectrometry. These results correlate with the B factors of available structures and the thermodynamic parameters associated with substrate binding, and suggest a higher level of conformational flexibility in the active site of EcMTAN. Our results implicate dynamics as an important factor in substrate selection in MTAN. [ABSTRACT FROM AUTHOR]

5′-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) is a dual substrate bacterial enzyme involved in S-adenosylmethionine (SAM) related quorum sensing pathways that regulates virulence in many bacterial species. MTANs from many bacteria are directly involved in the quorum sensing mechanism by regulating the synthesis of autoinducer molecules that are used by bacterial communities to communicate. In humans, 5′-methylthioadenosine phosphorylase (MTAP) is involved in polyamine biosynthesis as well as in purine and SAM salvage pathways and thus has been identified as an anticancer target. Previously we have described the synthesis and biological activity of several aza-C-nucleoside mimics with a sulfur atom at the 5′ position that are potent E. coliMTAN and human MTAP inhibitors. Because of the possibility that the sulfur may affect bioavailability, we were interested in synthesizing “sulfur-free” analogues. Herein we describe the preparation of a series of “sulfur-free” transition state analogue inhibitors of E. coliMTAN and human MTAP that have low nano- to picomolar dissociation constants and are potentially novel bacterial anti-infective and anticancer drug candidates. [ABSTRACT FROM AUTHOR]

Abstract: Genes encoding purine nucleoside phosphorylase (deo D), uridine phosphorylase (udp) and thimidine phosphorylase (deo A) from Escherichia coli BL21 were cloned and overexpressed in E. coli DH5α. The recombinant strains were employed to synthesize 2′-deoxyadenosine (dAR) and 6-methylpurine-2′-deoxyriboside (MePdR). Experimental parameters such as strains, temperature, pH, reagent concentration and cell mass were optimized. Under the optimal situation, 96% adenine was converted to dAR and 95% 6-methylpurine (MeP) was converted to MePdR in an hour, using 0.2‰ (dry wt./v) cell paste as biocatalyst. [Copyright &y& Elsevier]

Abstract: Epigenetic regulation through protein posttranslational modifications is essential in development and disease. Among the key chemical modifications is protein methylation carried out by protein methyltransferases (PMTs). Quantitative and sensitive PMT activity assays can provide valuable tools to investigate PMT functions. Here we developed an enzyme-coupled luminescence assay for S-adenosyl-l-methionine (AdoMet/SAM)-based PMTs. In this assay, S-adenosyl-l-homocystine (AdoHcy/SAH), the by-product of PMT-involved methylation, is sequentially converted to adenine, adenosine monophosphate, and then adenosine 5′-triphosphate (ATP) by 5′-methylthio-adenosine/AdoHcy nucleosidase (MTAN), adenine phosphoribosyl transferase (APRT), and pyruvate orthophosphate dikinase (PPDK), respectively. The resultant ATP can be readily quantified with a luciferin/luciferase kit. This assay is featured for its quantitative linear response to AdoHcy and the ultrasensitivity to 0.3pmol of AdoHcy. With this assay, the kinetic parameters of SET7/9 methylation were characterized and unambiguously support an ordered mechanism with AdoMet binding as the initial step, followed by the substrate binding and the rate-limiting methylation. The luminescence assay is also expected to be generally applicable to many other AdoMet-dependent enzymes. In addition, the mix-and-measure 96-/384-well format of our assay makes it suitable for automation and high throughput. Our enzyme-coupled luminescence assay, therefore, represents a convenient and ultrasensitive approach to examine methyltransferase activities and identify methyltransferase inhibitors. [Copyright &y& Elsevier]

Tyrosyl DNA-phosphodiesterase 1 (Tdp1) can disjoin peptides covalently bound to DNA. We assessed the role of Tdpl in nonhomologous end joining (NHEJ) and found that linear DNA molecules with 5' extensions showed a high frequency of misrepair in Δtdpl cells. The joining errOrs in Δtdp1 cells were predominantly 2-4 nucleotide insertions. Ends with 3' extensions or blunt ends did not show enhanced frequencies of errors, although Atdpl cells repaired blunt DNA ends with greater efficiency than WT cells. We found that insertions required Ku80 and DNA ligase IV. as well as polymerase IV. Our results show that yeast Tdpl is a component of the NHEJ pathway. We suggest that Tdplp 3' nucleosidase activity regulates the processing of DNA ends by generating a 3' phosphate, thereby restricting the ability of polymerases and other enzymes from acting at DNA ends. In support of this model, we found that overexpression of Tpp1, a yeast DNA 3' phosphatase, also leads to a higher frequency of insertions, suggesting that the generation of a 3' phosphate is a key step in Tdp1-mediated error prevention during NHEJ. [ABSTRACT FROM AUTHOR]

Summary: The gene Rcl encodes a deoxynucleoside 5′-monophosphate N-glycosidase that catalyzes the hydrolysis of the N-glycosidic bond of the nucleotide to give deoxyribose 5-phosphate and a nucleobase, preferentially a purine. This enzyme is over-expressed in several cancers, and its rate of expression is correlated to the degree of aggressiveness of tumors, which makes it a new and attractive therapeutic target. We describe here its structural characterization in the presence of two inhibitory substrate mimics. One of these ligands corresponds to the monophosphorylated form of acyclovir, which is used in the clinic. This study reveals an important ligand-induced stabilization of the dimer structure of the enzyme. The original structural features of Rcl will be helpful for designing new inhibitors. [Copyright &y& Elsevier]

Abstract: Members of the radical S-adenosylmethionine (AdoMet) superfamily reductively cleave AdoMet to generate the highly reactive 5′-deoxyadenosyl radical (DOA ) which initiates biological transformations by abstraction of a hydrogen atom. We demonstrate that three members of the family: biotin synthase (BioB), lipoyl synthase (LipA) and tyrosine lyase (ThiH) are inhibited in vitro by a combination of the products 5′-deoxyadenosine (DOA) and methionine. These results suggest the observed inhibition is a common feature of the radical AdoMet proteins that form DOA and methionine as products. Addition of 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) to BioB, LipA or ThiH activity assays removed the product inhibition by catalysing the hydrolysis of DOA and gave an increase in activity. [Copyright &y& Elsevier]

Abstract: 5′-Methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH) are important metabolites in all living organisms. Two similar nucleosidases for hydrolyzing MTA in Arabidopsis thaliana (AtMTAN1 and AtMTAN2) exist, but only AtMTAN2 shows markedly broad substrate specificity for hydrolysis of SAH. To examine the biochemical characteristics of AtMTAN2, it was over-expressed in Escherichia coli and purified to homogeneity. Spectroscopic assays confirm AtMTAN2 catalyzes MTA as well as SAH hydrolysis, compared to AtMTAN1 which only hydrolyzes MTA. In addition, crystal structure of the AtMTAN2 enzyme in complex with, adenine was determined at 2.9Å resolution. Finally, a structural comparison of AtMTAN2 performed with previously determined structures of AtMTAN1 and an E. coli homolog provides clues for the substrate specificity of MTA nucleosidases in A. thaliana. [Copyright &y& Elsevier]

5′-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) is a bacterial enzyme involved in S-adenosylmethionine–related quorum sensing pathways that induce bacterial pathogenesis factors. Transition state analogs MT-DADMe-Immucillin-A, EtT-DADMe-Immucillin-A and BuT-DADMe-Immucillin-A are slow-onset, tight-binding inhibitors of Vibrio cholerae MTAN (VcMTAN), with equilibrium dissociation constants of 73, 70 and 208 pM, respectively. Structural analysis of VcMTAN with BuT-DADMe-Immucillin-A revealed interactions contributing to the high affinity. We found that in V. cholerae cells, these compounds are potent MTAN inhibitors with IC50 values of 27, 31 and 6 nM for MT-, EtT- and BuT-DADMe-Immucillin-A, respectively; the compounds disrupt autoinducer production in a dose-dependent manner without affecting growth. MT- and BuT-DADMe-Immucillin-A also inhibited autoinducer-2 production in enterohemorrhagic Escherichia coli O157:H7 with IC50 values of 600 and 125 nM, respectively. BuT-DADMe-Immucillin-A inhibition of autoinducer-2 production in both strains persisted for several generations and caused reduction in biofilm formation. These results support MTAN's role in quorum sensing and its potential as a target for bacterial anti-infective drug design. [ABSTRACT FROM AUTHOR]

Nucleoside degradation and salvage are important metabolic pathways but hardly understood in plants. Recent work on human pathogenic protozoans like Leishmania and Trypanosoma substantiates an essential function of nucleosidase activity. Plant nucleosidases are related to those from protozoans and connect the pathways of nucleoside degradation and salvage. Here, we describe the cloning of such an enzyme from Arabidopsis thaliana, Uridine-Ribohydrolase 1 (URH1) and the characterization by complementation of a yeast mutant. Furthermore, URH1 was synthesized as a recombinant protein in Escherichia coli. The pure recombinant protein exhibited highest hydrolase activity for uridine, followed by inosine and adenosine, the corresponding Km values were 0.8, 1.4, and 0.7 mM, respectively. In addition, URH1 was able to cleave the cytokinin derivative isopentenyladenine-riboside. Promoter β-glucuronidase fusion studies revealed that URH1 is mainly transcribed in the vascular cells of roots and in root tips, guard cells, and pollen. Mutants expressing the Arabidopsis enzyme or the homolog from rice (Oryza sativa) exhibit resistance toward toxic fluorouridine, fluorouracil, and fluoroorotic acid, providing clear evidence for a pivotal function of URH1 as regulative in pyrimidine degradation. Moreover, mutants with increased and decreased nucleosidase activity are delayed in germination, indicating that this enzyme activity must be well balanced in the early phase of plant development. [ABSTRACT FROM AUTHOR]

Calcineurin B-like (CBL) proteins represent a unique family of calcium sensors in plant cells. Sensing the calcium signals elicited by a variety of abiotic stresses, CBLs transmit the information to a group of serine/threonine protein kinks (CBL-interacting protein kinases [CIPKs]), which are currently known as the sole targets of the CBL family. Here, we report that the CBL3 member of this family has a novel interaction partner in addition to the CIPK proteins. Extensive yeast two-hybrid screenings with CBL3 as bait identified an interesting Arabidopsis (Arabidopsis thaliana) cDNA clone (named AtMTAN, for 5'-methylthioadenosine nucleosidase), which encodes a polypeptide similar to EcMTAN from Escherichia coli. Deletion analyses showed that CBL3 utilizes the different structural modules to interact with its distinct target proteins, CIPKs and AtMTAN. In vitro and in vivo analyses verified that CBL3 and AtMTAN physically associate only in the presence of Ca2+. In addition, we empirically demonstrated that the AtMTAN protein indeed possesses the MTAN activity, which can be inhibited specifically by Ca2+-bound CBL3. Overall, these findings suggest that the CBL family members can relay the calcium signals in more diverse ways than previously thought. We also discuss a possible mechanism by which the CBL3-mediated calcium signaling regulates the biosynthesis of ethylene and polyamines, which are Involved in plant growth and development as well as various stress responses. [ABSTRACT FROM AUTHOR]

Abstract: 5′-Methylthioadenosine (MTA)/S-adenosylhomocysteine (SAH) nucleosidase (MTAN) is essential for cellular metabolism and development in many bacterial species. While the enzyme is found in plants, plant MTANs appear to select for MTA preferentially, with little or no affinity for SAH. To understand what determines substrate specificity in this enzyme, MTAN homologues from Arabidopsis thaliana (AtMTAN1 and AtMTAN2, which are referred to as AtMTN1 and AtMTN2 in the plant literature) have been characterized kinetically. While both homologues hydrolyze MTA with comparable kinetic parameters, only AtMTAN2 shows activity towards SAH. AtMTAN2 also has higher catalytic activity towards other substrate analogues with longer 5′-substituents. The structures of apo AtMTAN1 and its complexes with the substrate- and transition-state-analogues, 5′-methylthiotubercidin and formycin A, respectively, have been determined at 2.0–1.8 Å resolution. A homology model of AtMTAN2 was generated using the AtMTAN1 structures. Comparison of the AtMTAN1 and AtMTAN2 structures reveals that only three residues in the active site differ between the two enzymes. Our analysis suggests that two of these residues, Leu181/Met168 and Phe148/Leu135 in AtMTAN1/AtMTAN2, likely account for the divergence in specificity of the enzymes. Comparison of the AtMTAN1 and available Escherichia coli MTAN (EcMTAN) structures suggests that a combination of differences in the 5′-alkylthio binding region and reduced conformational flexibility in the AtMTAN1 active site likely contribute to its reduced efficiency in binding substrate analogues with longer 5′-substituents. In addition, in contrast to EcMTAN, the active site of AtMTAN1 remains solvated in its ligand-bound forms. As the apparent pK a of an amino acid depends on its local environment, the putative catalytic acid Asp225 in AtMTAN1 may not be protonated at physiological pH and this suggests the transition state of AtMTAN1, like human MTA phosphorylase and Streptococcus pneumoniae MTAN, may be different from that found in EcMTAN. [Copyright &y& Elsevier]

The yeast pathogen Candida glabrata is a nicotinamide adenine dinucleotide (NAD+) auxotroph and its growth depends on the environmental supply of vitamin precursors of NAD+. C. glabrata salvage pathways defined in this article allow NAD+ to be synthesized from three compounds – nicotinic acid (NA), nicotinamide (NAM) and nicotinamide riboside (NR). NA is salvaged through a functional Preiss–Handler pathway. NAM is first converted to NA by nicotinamidase and then salvaged by the Preiss–Handler pathway. Salvage of NR in C. glabrata occurs via two routes. The first, in which NR is phosphorylated by the NR kinase Nrk1, is independent of the Preiss–Handler pathway. The second is a novel pathway in which NR is degraded by the nucleosidases Pnp1 and Urh1, with a minor role for Meu1, and ultimately converted to NAD+ via the nicotinamidase Pnc1 and the Preiss–Handler pathway. Using C. glabrata mutants whose growth depends exclusively on the external NA or NR supply, we also show that C. glabrata utilizes NR and to a lesser extent NA as NAD+ sources during disseminated infection. [ABSTRACT FROM AUTHOR]

Methylthioadenosine (MTA) is released as a by-product of S-adenosylmethionine (AdoMet)-dependent reactions central to ethylene, polyamine, or phytosiderophore biosynthesis. MTA is hydrolysed by methylthioadenosine nucleosidase (MTN; EC 3.2.2.16) into adenine and methylthioribose which is processed through the methionine (Met) cycle to produce a new molecule of AdoMet. In deepwater rice, submergence enhances ethylene biosynthesis, and ethylene in turn influences the methionine cycle through positive feedback regulation of the acireductone dioxygenase gene OsARD1. In rice, MTN is encoded by a single gene designated OsMTN. Recombinant OsMTN enzyme had a KM for MTA of 2.1 mM and accepted a wide array of 5′ substitutions of the substrate. OsMTN also metabolized S-adenosylhomocysteine (AdoHcy) with 15.9% the rate of MTA. OsMTN transcripts and OsMTN-specific activity increased slowly and in parallel upon submergence, indicating that regulation occurred mainly at the transcriptional level. Neither ethylene, MTA, nor Met regulated OsMTN expression. Analysis of steady-state metabolite levels showed that MTN activity was sufficiently high to prevent Met and AdoMet depletion during long-term ethylene biosynthesis. [ABSTRACT FROM PUBLISHER]

Kinetic isotope effects (KIEs) and computer modeling are used to approximate the transition state of S. pneumoniae 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN). Experimental KIEs were measured and corrected for a small forward commitment factor. Intrinsic KIEs were obtained for [1′-³H], (1′-14C], [2′-³H], [4′-³H], [5′-³H2], [9-15N] and [Me-³H3] MTAs. The intrinsic KlEs suggest an SN1 transition state with no covalent participation of the adenine or the water nucleophile. The transition state was modeled as a stable ribooxacarbenium ion intermediate and was constrained to fit the intrinsic KIEs. The isotope effects predicted a 3-endo conformation for the ribosyl oxacarbenium-ion corresponding to H1′-C1′-C2′-H2′ dihedral angle of 70°. Ab initio Hartree-Fock and DFT calculations were performed to study the effect of polarization of ribosyl hydroxyls, torsional angles, and the effect of base orientation on isotope effects. Calculations suggest that the 4′-³H KIE arises from hyperconjugation between the lonepair (np) of O4′ and the σ* (C4′-H4′) antibonding orbital owing to polarization of the 3′-hydroxyl by Glu174. A [methyl-³H3] KIE is due to hyperconjugation between np of sulfur and σ* of methyl C-H bonds. The van der Waal contacts increase the 1′-³H KIE because of induced dipole-dipole interactions. The 1′-³H KIE is also influenced by the torsion angles of adjacent atoms and by polarization of the 2′-hydroxyl. Changing the virtual solvent (dielectric constant) does not influence the isotope effects. Unlike most N-ribosyltransferases, N7 of the leaving group adenine is not protonated at the transition state of S. pneumoriiae MTAN. This feature differentiates the S. pneumoniae and E. coli transition states and explains the 10³-fold decrease in the catalytic efficiency of S. pneumoniae MTAN relative to that from E. coli. [ABSTRACT FROM AUTHOR]

Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) catalyzes reactions linked to polyamine metabolism, quorum sensing pathways, methylation reactions, and adenine salvage. It is a candidate target for antimicrobial drug design. Kinetic isotope effects (KIEs) were measured on the MTAN- catalyzed hydrolysis of 5'-methylthioadenosine (MTA) to determine the transition state structure. KIEs measured at pH 7.5 were near unity due to the large forward commitment to catalysis. Intrinsic KIEs were expressed by increasing the pH to 8.5. Intrinsic KIEs from MTAs labeled at 1'-³H, 1'-14C, 2'-³H, 4'-³H, 5'-³H, 9-15N, and Me-³H3 were 1.160 ± 0.004, 1.004 ± 0.003, 1.044 ± 0.004, 1.015 ± 0.002, 1.010 ± 0.002, 1.018 ± 0.006, and 1.051 ± 0.002, respectively. The large 1'-³H and small 1'-14C KIEs indicate that the Escherichia coli MTAN reaction undergoes a dissociative (DN*AN) (SN1) mechanism with little involvement of the leaving group or participation of the attacking nucleophile at the transition state, causing the transition state to have significant ribooxacarbenium ion character. A transition state constrained to match the intrinsic KIEs was located with density functional theory [B3LYP/6-31G(d,p)]. The leaving group (N9) is predicted to be 3.0 Å from the anomeric carbon. The small β-secondary 2'-³H KIE of 1.044 corresponds to a modest 3'-endo conformation for ribose and a H1'-C1'-C2'H2' dihedral angle of 53° at the transition state. Natural bond orbital analysis of the substrate and the transition state suggests that the 4'-³H KIE is due to hyperconjugation between the lone pair (np) of O3' and the antibonding (σ*) orbital of the C4'-H4' group, and the methyl-³H3 ME is due to hyperconjugation between the np of sulfur and the σ* of methyl C–H bonds. Transition state analogues that resemble this transition state structure are powerful inhibitors, and their molecular electrostatic potential maps closely resemble that of the transition state. [ABSTRACT FROM AUTHOR]

5'-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) is important in a number of cellular functions such as polyamine biosynthesis, methionine salvaging, biological methylation, and quorum sensing. The nucleosidase is found in many microbes but not in mammalian systems, thus making MTAN a broad-spectrum antimicrobial drug target. Substrate binding and catalytic residues were identified from the crystal structure of MTAN complexed with 5'-methylthiotubercidin [Lee, J. E., Cornell, K. A., Riscoe, M. K. and Howell, P. L. (2003) J. Biol. Chem. 278 (10) 8761-8770]. The roles of active site residues Met9, Glu12, Ile50, Ser76, Val102, Phe105, Tyr107, Phe151, Met173, Glu174, Arg193, Ser196, Asp 197, and Phe207 have been investigated by site-directed mutagenesis and steady-state kinetics. Mutagenesis of residues Glu12, Glu 174, and Asp 197 completely abolished activity. The location of Asp 197 and Glu12 in the active site is consistent with their having a direct role in enzyme catalysis. Glu 174 is suggested to be involved in catalysis by stabilizing the transition state positive charge at the O3', C2', and C3' atoms and by polarizing the 3'-hydroxyl to aid in the flow of electrons to the electron withdrawing purine base. This represents the first indication of the importance of the 3'-hydroxyl in the stabilization of the transition state. Furthermore, mutation of Arg 193 to alanine shows that the nucleophilic water is able to direct its attack without assistance from the enzyme. This mutagenesis study has allowed a reevaluation of the catalytic mechanism. [ABSTRACT FROM AUTHOR]

Immucillin and DADMe. Immucillin inhibitors are tight binding transition state mimics of purine nucleoside phosphorylases (PNP). 5'-Methylthioadenosine/Sadenosylhomocysteine nucleosidase (MTAN) is proposed to form a similar transition state structure as PNP. The companion paper describes modifications of the Immucillin and DADMe-Immucillin inhibitors to better match transition state features of MTAN and have led to 5'-thio aromatic substitutions that extend the inhibition constants to the femtomolar range (Singh, V., Evans, G. B., Lenz, D. H., Mason, J., Clinch, K., Mee, S., Painter, G. F., Tyler, P. C., Furneaux, R. H., Lee, J. E., Howell, P. L., and Schramm, V. L. (2005) J. Biol. Chem. 280, 18265-18273). 5'-Methylthio-Immucillin A (MTImmA) and 5'-methylthio-DADMe-Immucillin A (MTDADMe-ImmA) exhibit slow-onset inhibition with Ki* of 77 and 2 pM, respectively, and were selected for structural analysis as the parent compounds of each class of transition state analogue. The crystal structures of Escherichia coli MTAN complexed with MT-ImmA and MT-DADMe-ImmA were determined to 2.2 Å resolution and compared with the existing MTAN inhibitor complexes. These MTAN-transition state complexes are among the tightest binding enzyme-ligand complexes ever described and analysis of their mode of binding provides extraordinary insight into the structural basis for their affinity. The MTAN-MT-ImmA complex reveals the presence of a new ion pair between the 4'-iminoribitol atom and the nucleophilic water (WAT3) that captures key features of the transition state. Similarly, in the MTAN-MT-DADMe-ImmA complex a favorab1e hydrogen bond or ion pair interaction between the cationic 1'-pyrrolidine atom and WAT3 is crucial for tight affinity. Distance ana1ysis of the nucleophile and 1eaving group show that MT-ImmA is a mimic of an early transition state, while MT-DADMe-ImmA is a better mimic of the highly dissociated transition state of E. coli MTAN. [ABSTRACT FROM AUTHOR]

Escherichia coli 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) hydrolyzes its substrates to form adenine and 5-methylthioribose (MTR) or S-ribosylhomocysteine (SRH). 5'-Methylthioadenosine (MTA) is a by-product of polyamine synthesis and SRH is a precursor to the biosynthesis of one or more quorum sensing autoinducer molecules. MTAN is therefore involved in quorum sensing, recycling MTA from the polyamine pathway via adenine phosphoribosyltransferase and recycling MTR to methionine. Hydrolysis of MTA by E. coli MTAN involves a highly dissociative transition state with ribooxacarbenium ion character. Iminoribitol mimics of MTA at the transition state of MTAN were synthesized and tested as inhibitots. 5'-Methylthio-Immucillin-A (MT-ImmA) is a slowonset tight-binding inhibitor giving a dissociation constant (Ki*) of 77 pM. Substitution of the methylthio group with a p-Cl-phenylthio group gives a more powerful inhibitor with a dissociation constant of 2 pM. DADMeImmucillins are better inhibitors of E. coli MTAN, since they are more closely related to the highly dissociative nature of the transition state. MT-DADMe-Immucillin-A binds with a Ki* value of 2 pM. Replacing the 5'-methyl group with other hydrophobic groups gave 17 transition state analogue inhibitors with dissociation constants from 10-12 to 10-14 M. The most powerful inhibitor was 5'-p-Cl-phenylthio-DADMe-Immucillin-A (pCIPhTDADMe-ImmA) with a Ki* value of 47 fM (47 x 10-15 M). These are among the most powerful non-covalent inhibitors reported for any enzyme, binding 9-91 million times tighter than the MTA and SAH substrates, respectively. The inhibitory potential of these transition state analogue inhibitors supports a transition state structure closely resembling a fully dissociated ribooxacarbenium ion. Powerful inhibitors of MTAN are candidates to disrupt key bacterial pathways including methylation, polyamine synthesis, methionine salvage, and quorum sensing. The accompanying article reports crystal structures of MTAN with these analogues. [ABSTRACT FROM AUTHOR]