methylthioadenosine/S-adenosyl Homocysteine Nucleosidase Research Articles

Klebsiella Pneumoniae Volatile Organic Compounds (VOCs) Protect Artemia salina from Fish Pathogen Aeromonas sp.: A Combined In Vitro, In Vivo, and In Silico Approach

Antibiotic resistance is an alarming threat all over the world, and the biofilm formation efficacy of bacteria is making the situation worse. The antagonistic efficacy of Klebsiella pneumoniae against one of the known fish pathogens, Aeromonas sp., is examined in this study. Moreover, Aeromonas sp.'s biofilm formation ability and in vivo pathogenicity on Artemia salina are also justified here. Firstly, six selected bacterial strains were used to obtain antimicrobial compounds against this pathogenic strain. Among those, Klebsiella pneumoniae, another pathogenic bacterium, surprisingly demonstrated remarkable antagonistic activity against Aeromonas sp. in both in vitro and in vivo assays. The biofilm distrusting potentiality of Klebsiella pneumoniae's cell-free supernatants (CFSs) was likewise found to be around 56%. Furthermore, the volatile compounds of Klebsiella pneumoniae were identified by GC-MS in order to explore compounds with antibacterial efficacy against Aeromonas sp. through an in silico study, where 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) (PDB: 5B7P) was chosen as a target protein for its unique characteristics and pathogenicity. Several volatile compounds, such as oxime- methoxy-phenyl-, fluoren-9-ol, 3,6-dimethoxy-9-(2-phenylethynyl)-, and 2H-indol-2-one, 1,3-dihydro- showed a strong binding affinity, with free energy of -6.7, -7.1, and -6.4 Kcal/mol, respectively, in complexes with the protein MTAN. Moreover, the root-mean-square deviation, solvent-accessible surface area, radius of gyration, root-mean-square fluctuations, and hydrogen bonds were used to ensure the binding stability of the docked complexes in the atomistic simulation. Thus, Klebsiella pneumoniae and its potential compounds can be employed as an alternative to antibiotics for aquaculture, demonstrating their effectiveness in suppressing Aeromonas sp.

Precise Positioning of Water Is Critical for Hydrolysis Catalyzed by 5'-Methylthioadenosine Nucleosidase.

Enzyme-catalyzed hydrolysis is a fundamental chemical transformation involved in many essential metabolic processes. The enzyme 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) catalyzes the hydrolysis of adenosine-containing metabolites in cysteine and methionine metabolism. Although MTAN enzymes contain highly similar active site architecture and generally follow a dissociative (DN*AN) reaction mechanism, substantial differences in reaction rates and chemical transition state structures have been reported. To understand how subtle changes in sequence and structure give rise to differences in chemistry between homologous enzymes, we have probed the reaction coordinates of two MTAN enzymes using quantum mechanical/molecular mechanical and molecular dynamics simulations combined with experimental methods. We show that the transition state structure and energy are significantly affected by the recruitment and positioning of the catalytic water molecule and that subtle differences in the noncatalytic active site residues alter the environment of the catalytic water, leading to changes in the reaction coordinate and observed reaction rate.

5-Fluorouracil blocks quorum-sensing of biofilm-embedded methicillin-resistant Staphylococcus aureus in mice.

Antibiotic-resistant pathogens often escape antimicrobial treatment by forming protective biofilms in response to quorum-sensing communication via diffusible autoinducers. Biofilm formation by the nosocomial pathogen methicillin-resistant Staphylococcus aureus (MRSA) is triggered by the quorum-sensor autoinducer-2 (AI-2), whose biosynthesis is mediated by methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) and S-ribosylhomocysteine lyase (LuxS). Here, we present a high-throughput screening platform for small-molecular inhibitors of either enzyme. This platform employs a cell-based assay to report non-toxic, bioavailable and cell-penetrating inhibitors of AI-2 production, utilizing engineered human cells programmed to constitutively secrete AI-2 by tapping into the endogenous methylation cycle via ectopic expression of codon-optimized MTAN and LuxS. Screening of a library of over 5000 commercial compounds yielded 66 hits, including the FDA-licensed cytostatic anti-cancer drug 5-fluorouracil (5-FU). Secondary screening and validation studies showed that 5-FU is a potent quorum-quencher, inhibiting AI-2 production and release by MRSA, Staphylococcus epidermidis, Escherichia coli and Vibrio harveyi. 5-FU efficiently reduced adherence and blocked biofilm formation of MRSA in vitro at an order-of-magnitude-lower concentration than that clinically relevant for anti-cancer therapy. Furthermore, 5-FU reestablished antibiotic susceptibility and enabled daptomycin-mediated prevention and clearance of MRSA infection in a mouse model of human implant-associated infection.

Synthesis, Characterization and in vitro Antimicrobial Evaluation of Chalconeimine Derivatives as Potential Inhibitors against Enzymes Produced from S. aureus: A Computational Approach

(Z)-1-(Benzo[d][1,3]dioxol-5-yl)-3-(4-(difluoromethoxy)-3-hydroxyphenyl)prop-2-en-1-one hydrazone derivatives pronounced in this manuscript represents a new collection of antibacterial agents in addition to the DNA gyrase inhibitors. Efforts had been made to synthesize those chalcone-hydrazone derivatives (4a-e) in good yields. The literature survey confirms that nano-ZnO as heterogeneous catalyst has obtained big interest because of its ecofriendly nature and has been explored as a effective catalyst for several organic ameliorations. Subsequently, induced by way of these observations and in continuation to our interest in organic synthesis with using nanocatalyst. in vitro Antibacterial activity has been evaluated towards Gram-positive and Gram-negative bacterial strains for all compounds. So one can discover the affinity to bacterial proteins docking have a look at have been carried out for 5 synthesized derivatives, antibiotic drug and co-crystallized ligands with special mechanism of action DNA gyrase B and methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) the usage of AutoDock 4.

Selective Inhibitors of Helicobacter pylori Methylthioadenosine Nucleosidase and Human Methylthioadenosine Phosphorylase.

Bacterial 5'-methylthioadenosine/ S-adenosylhomocysteine nucleosidase (MTAN) hydrolyzes adenine from its substrates to form S-methyl-5-thioribose and S-ribosyl-l-homocysteine. MTANs are involved in quorum sensing, menaquinone synthesis, and 5'-methylthioadenosine recycling to S-adenosylmethionine. Helicobacter pylori uses MTAN in its unusual menaquinone pathway, making H. pylori MTAN a target for antibiotic development. Human 5'-methylthioadenosine phosphorylase (MTAP), a reported anticancer target, catalyzes phosphorolysis of 5'-methylthioadenosine to salvage S-adenosylmethionine. Transition-state analogues designed for HpMTAN and MTAP show significant overlap in specificity. Fifteen unique transition-state analogues are described here and are used to explore inhibitor specificity. Several analogues of HpMTAN bind in the picomolar range while inhibiting human MTAP with orders of magnitude weaker affinity. Structural analysis of HpMTAN shows inhibitors extending through a hydrophobic channel to the protein surface. The more enclosed catalytic sites of human MTAP require the inhibitors to adopt a folded structure, displacing the phosphate nucleophile from the catalytic site.

Structural and Functional Analyses of Periplasmic 5'-Methylthioadenosine/S-Adenosylhomocysteine Nucleosidase from Aeromonas hydrophila.

The Gram-negative, rod-shaped bacterium Aeromonas hydrophila has two multifunctional 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) enzymes, MtaN-1 and MtaN-2, that differ from those in other bacteria. These proteins are essential for several metabolic pathways, including biological methylation, polyamine biosynthesis, methionine recycling, and bacterial quorum sensing. To gain insight into how these two proteins function, we determined four high-resolution crystal structures of MtaN-1 in its apo form and in complex with the substrates S-adenosyl-l-homocysteine, 5'-methylthioadenosine, and 5'-deoxyadenosine. We found that the domain structures were generally similar, although slight differences were evident. The crystal structure demonstrates that AhMtaN-1 has an extension of the binding pocket and revealed that a tryptophan in the active site (Trp199) may play a major role in substrate binding, unlike in other MTAN proteins. Mutation of the Trp199 residue completely abolished the enzyme activity. Trp199 was identified as an active site residue that is essential for catalysis. Furthermore, biochemical characterization of AhMtaN-1 and AhMtaN-2 demonstrated that AhMtaN-1 exhibits inherent trypsin resistance that is higher than that of AhMtaN-2. Additionally, the thermally unfolded AhMtaN-2 protein is capable of refolding into active forms, whereas the thermally unfolded AhMtaN-1 protein does not have this ability. Examining the different biochemical characteristics related to the functional roles of AhMtaN-1 and AhMtaN-2 would be interesting. Indeed, the biochemical characterization of these structural features would provide a structural basis for the design of new antibiotics against A. hydrophila.

Transition State Structure and Inhibition of Rv0091, a 5'-Deoxyadenosine/5'-methylthioadenosine Nucleosidase from Mycobacterium tuberculosis.

5'-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) is a bacterial enzyme that catalyzes the hydrolysis of the N-ribosidic bond in 5'-methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH). MTAN activity has been linked to quorum sensing pathways, polyamine biosynthesis, and adenine salvage. Previously, the coding sequence of Rv0091 was annotated as a putative MTAN in Mycobacterium tuberculosis. Rv0091 was expressed in Escherichia coli, purified to homogeneity, and shown to be a homodimer, consistent with MTANs from other microorganisms. Substrate specificity for Rv0091 gave a preference for 5'-deoxyadenosine relative to MTA or SAH. Intrinsic kinetic isotope effects (KIEs) for the hydrolysis of [1'-(3)H], [1'-(14)C], [5'-(3)H2], [9-(15)N], and [7-(15)N]MTA were determined to be 1.207, 1.038, 0.998, 1.021, and 0.998, respectively. A model for the transition state structure of Rv0091 was determined by matching KIE values predicted via quantum chemical calculations to the intrinsic KIEs. The transition state shows a substantial loss of C1'-N9 bond order, well-developed oxocarbenium character of the ribosyl ring, and weak participation of the water nucleophile. Electrostatic potential surface maps for the Rv0091 transition state structure show similarity to DADMe-immucillin transition state analogues. DADMe-immucillin transition state analogues showed strong inhibition of Rv0091, with the most potent inhibitor (5'-hexylthio-DADMe-immucillinA) displaying a Ki value of 87 pM.

New Antibiotic Candidates against Helicobacter pylori.

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.

Molecular dynamics study of the effect of active site protonation on Helicobacter pylori 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase.

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 K2 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.

Crystal structure and biochemical studies of Brucella melitensis 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase

The prokaryotic 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) catalyzes the irreversible cleavage of the glycosidic bond in 5′-methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH), a process that plays a key role in several metabolic pathways. Its absence in all mammalian species has implicated this enzyme as a promising target for antimicrobial drug design. Here, we report the crystal structure of BmMTAN in complex with its product adenine at a resolution of 2.6Å determined by single-wavelength anomalous dispersion method. 11 key residues were mutated for kinetic characterization. Mutations of Tyr134 and Met144 resulted in the largest overall increase in Km, whereas mutagenesis of residues Glu18, Glu145 and Asp168 completely abolished activity. Glu145 and Asp168 were identified as active site residues essential for catalysis. The catalytic mechanism and implications of this structure for broad-based antibiotic design are discussed.

Enzyme‐Substrate Interactions in the Helicobacter pylori 5′‐Methylthioadenosine/S‐adenosylhomocysteine Nucleosidase

5′‐methylthioadenosine/S‐adenosylhomocysteine nucleosidase (MTAN) is a multi‐functional bacterial enzyme that catalyzes the hydrolysis of the N‐glycosidic bond of many adenosine‐based metabolites. This hydrolytic activity places MTAN at the hub of at least seven different metabolic pathways, which includes the purine salvage pathway, the methionine salvage pathway, various S‐adenosylmethionine (SAM)‐dependent methylation pathways, polyamine biosynthesis, the production of quorum sensing molecules and menaquinone biosynthesis. The bacterial species such as Campylobacter and Helicobacter are dependent on MTAN for menaquinone synthesis, an essential metabolite for bacterial viability. As MTAN is absent in humans but essential for bacteria like Helicobacter pylori, it is an excellent target for the development of new treatments for Helicobacter infections. To structurally characterize the interactions between MTAN and its various substrates, complexes of an inactive mutant of the Helicobacter pylori MTAN with two known substrates, 5′‐methylthioadenosine (MTA) and S‐adenosylhomocysteine (SAH) were formed and crystallized. The crystal structures of mutant MTAN with SAH and MTA bound in its active site were solved to 1.2 and 1.6 Å, respectively. The MTAN‐SAH co‐crystal structure represents the first visualization of interactions between the homocysteine moiety of SAH and the 5′‐alkylthiol‐binding sub‐site of the MTAN active site. The co‐crystal structure of wild‐type MTAN with products, adenine and S‐ribosylhomocysteine, was also determined to 1.54 Å resolution. The similarities and differences in these three structures highlight features that can be exploited to design H. pylori specific drugs.

Substrate preference of 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase inBurkholderia thailandensis

5'-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) plays crucial roles in the production of autoinducers and methionine metabolism. Putative genes encoding MTAN and AdoHcyase from Burkholderia thailandensis were cloned and characterized. The K(m) values of MTAN for 5'-methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH) were 19 and 58μM, respectively. The catalytic efficiency of MTAN for SAH was only 0.004% of the value for MTA, indicating an almost complete substrate preference of MTAN for MTA. The results of autoinducer-2 assay of B.thailandensis and recombinants indicated that LuxS enzyme activity was lacking in Burkholderia species. Instead, AdoHcyase hydrolysed SAH directly to homocysteine and adenosine in the activated methyl cycle. Meanwhile, the K(m) value of AdoHcyase for SAH was determined to be 40μM. Sequence analysis revealed that MTAN had much higher diversity than AdoHcyase, which likely contributes to its substrate preference for MTA. Furthermore, the phylogenetic tree of MTAN sequences revealed that LuxS(+) bacteria could be discriminated from LuxS(-) bacteria. These results suggested that the substrate preference of MTAN for MTA and SAH degradation pathway evolved with the bacterial-activated methyl cycle.

Crystal Structures of the Helicobacter pylori MTAN Enzyme Reveal Specific Interactions between S-Adenosylhomocysteine and the 5′-Alkylthio Binding Subsite

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.

Femtomolar Inhibitors Bind to 5′-Methylthioadenosine Nucleosidases with Favorable Enthalpy and Entropy

5'-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) catalyzes the hydrolytic cleavage of adenine from methylthioadenosine (MTA). Inhibitor design and synthesis informed by transition state analysis have developed femtomolar inhibitors for MTANs, among the most powerful known noncovalent enzyme inhibitors. Thermodynamic analyses of the inhibitor binding reveals a combination of highly favorable contributions from enthalpic (-24.7 to -4.0 kcal mol(-1)) and entropic (-10.0 to 6.4 kcal mol(-1)) interactions. Inhibitor binding to similar MTANs from different bacterial species gave distinct energetic contributions from similar catalytic sites. Thus, binding of four transition state analogues to EcMTAN and SeMTAN is driven primarily by enthalpy, while binding to VcMTAN is driven primarily by entropy. Human MTA phosphorylase (hMTAP) has a transition state structure closely related to that of the bacterial MTANs, and it binds tightly to some of the same transition state analogues. However, the thermodynamic signature of binding of an inhibitor to hMTAP differs completely from that with MTANs. We conclude that factors other than first-sphere catalytic residue contacts contribute to binding of inhibitors because the thermodynamic signature differs between bacterial species of the same enzyme.

Structural features for substrate recognition by bacterial 5′‐methylthioadenosine nucleosidase

The bacterial enzyme 5′‐methylthioadenosine/S‐adenosylhomocysteine nucleosidase (MTAN) is a multifunctional enzyme that catalyzes the hydrolysis of the N‐ribosidic bond of at least 3 different adenosine‐based metabolites: S‐adenosylhomocysteine (SAH), 5′‐deoxyadenosine and 5′‐methylthioadenosine (MTA). These activities place the enzyme at the hub of 5 fundamental bacterial metabolic pathways: S‐adenosylmethionine (SAM) utilization, the purine salvage pathway, the methionine salvage pathway, the SAM radical pathways and autoinducer‐2 biosynthesis. The last pathway is important in synthesizing diffusible compounds used in bacterial quorum sensing, which affects biofilm formation in many pathogenic bacteria. 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. To gain information regarding these interactions, we have solved crystal structures of an inactive mutant form of Helicobacter pylori MTAN bound to MTA and SAH to 1.65 and 1.2 Å, respectively. These structures identify specific interactions between the homocysteine moiety and the 5′‐alkylthiol binding site of the enzyme that appear to be conserved in campylobacter but not across all bacterial MTANs. Comparison of the MTA and SAH structures also shows that the localized structure of 5′‐alkylthiol binding site is identical without regard for which substrate is bound. This suggests that HpMTAN binding of the homocysteine moiety contributes little or no entropic penalty to this binding interaction and that these new data may promote development of species‐specific inhibitors for MTAN that affect biofilm formation.

Measuring a Stabilization Constant Between Two Bio-Molecules using Atomic Force Microscopy

Stabilization force constants are known to play an important role in biomolecular functions that carry out delicate structural conformation changes along a reaction coordinate during bio-molecular activation. Atomic force microscopy (AFM) has been used as a tool for probing protein-ligand interactions at the single molecular level. We developed a method that converts AFM force-distance curves into intermolecular force-distance curves. This method was applied to a model enzyme-inhibitor system of 5’ methylthioadenosine/S-adenosylhomocysteine nucleo-sidase (MTN) and its transition state analogue homocysteinyl Immucillin A (HIA).

Enzyme–ligand interactions that drive active site rearrangements in the Helicobacter pylori 5′‐methylthioadenosine/S‐adenosylhomocysteine nucleosidase

The bacterial enzyme 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) plays a central role in three essential metabolic pathways in bacteria: methionine salvage, purine salvage, and polyamine biosynthesis. Recently, its role in the pathway that leads to the production of autoinducer II, an important component in quorum-sensing, has garnered much interest. Because of this variety of roles, MTAN is an attractive target for developing new classes of inhibitors that influence bacterial virulence and biofilm formation. To gain insight toward the development of new classes of MTAN inhibitors, the interactions between the Helicobacter pylori-encoded MTAN and its substrates and substrate analogs were probed using X-ray crystallography. The structures of MTAN, an MTAN-Formycin A complex, and an adenine bound form were solved by molecular replacement and refined to 1.7, 1.8, and 1.6 Å, respectively. The ribose-binding site in the MTAN and MTAN-adenine cocrystal structures contain a tris[hydroxymethyl]aminomethane molecule that stabilizes the closed form of the enzyme and displaces a nucleophilic water molecule necessary for catalysis. This research gives insight to the interactions between MTAN and bound ligands that promote closing of the enzyme active site and highlights the potential for designing new classes of MTAN inhibitors using a link/grow or ligand assembly development strategy based on the described H. pylori MTAN crystal structures.

Design and Synthesis of Potent “Sulfur-Free” Transition State Analogue Inhibitors of 5′-Methylthioadenosine Nucleosidase and 5′-Methylthioadenosine Phosphorylase

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. coli MTAN 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. coli MTAN and human MTAP that have low nano- to picomolar dissociation constants and are potentially novel bacterial anti-infective and anticancer drug candidates.

Mechanism of substrate specificity in 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidases

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 thalianaAtMTAN1 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.

Product inhibition in the radical S-adenosylmethionine family

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.