Transition state structure of 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase from Escherichia coli and its similarity to transition state analogues

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'-(3)H, 1'-(14)C, 2'-(3)H, 4'-(3)H, 5'-(3)H, 9-(15)N, and Me-(3)H(3) 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'-(3)H and small 1'-(14)C KIEs indicate that the Escherichia coli MTAN reaction undergoes a dissociative (D(N)A(N)) (S(N)1) 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 A from the anomeric carbon. The small beta-secondary 2'-(3)H KIE of 1.044 corresponds to a modest 3'-endo conformation for ribose and a H1'-C1'-C2'-H2' dihedral angle of 53 degrees at the transition state. Natural bond orbital analysis of the substrate and the transition state suggests that the 4'-(3)H KIE is due to hyperconjugation between the lone pair (n(p)) of O3' and the antibonding (sigma) orbital of the C4'-H4' group, and the methyl-(3)H(3) KIE is due to hyperconjugation between the n(p) of sulfur and the sigma 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.     

Transition state analysis for human and Plasmodium falciparum purine nucleoside phosphorylases

Recent studies have shown that Plasmodium falciparum is sensitive to a purine salvage block at purine nucleoside phosphorylase (PNP) and that human PNP is a target for T-cell proliferative diseases. Specific tight-binding inhibitors might be designed on the basis of specific PNP transition state structures. Kinetic isotope effects (KIEs) were measured for arsenolysis of inosine catalyzed by P. falciparum and human purine nucleoside phosphorylases. Intrinsic KIEs from [1'-(3)H]-, [2'-(3)H]-, [1'-(14)C]-, [9-(15)N]-, and [5'-(3)H]inosines were 1.184 +/- 0.004, 1.031 +/- 0.004, 1.002 +/- 0.006, 1.029 +/- 0.006, and 1.062 +/- 0.002 for the human enzyme and 1.116 +/- 0.007, 1.036 +/- 0.003, 0.996 +/- 0.006, 1.019 +/- 0.005, and 1.064 +/- 0.003 for P. falciparum PNPs, respectively. Analysis of KIEs indicated a highly dissociative D(N)A(N) (S(N)1) stepwise mechanism with very little leaving group involvement. The near-unity 1'-(14)C KIEs for both human and P. falciparum PNP agree with the theoretical value for a 1'-(14)C equilibrium isotope effect for oxacarbenium ion formation when computed at the B1LYP/6-31G(d) level of theory. The 9-(15)N KIE for human PNP is also in agreement with theory for equilibrium formation of hypoxanthine and oxacarbenium ion at this level of theory. The 9-(15)N KIE for P. falciparum PNP shows a constrained vibrational environment around N9 at the transition state. A relatively small beta-secondary 2'-(3)H KIE for both enzymes indicates a 3'-endo conformation for ribose and relatively weak hyperconjugation at the transition state. The large 5'-(3)H KIE reveals substantial distortion at the 5'-hydroxymethyl group which causes loosening of the C5'-H5' bonds during the reaction coordinate.     

Inhibition of ricin A-chain with pyrrolidine mimics of the oxacarbenium ion transition state

Ricin A-chain (RTA) catalyzes the hydrolytic depurination of a specific adenosine at position 4324 of 28S rRNA. Kinetic isotope effects on the hydrolysis of a small 10mer stem-tetraloop oligonucleotide substrate established the mechanism of the reaction as D(N)*A(N), involving an oxacarbenium ion intermediate in a highly dissociative transition state. An inhibitor with a protonated 1,4-dideoxy-1,4-imino-D-ribitol moiety, a 4-azasugar mimic, at the depurination site in the tetraloop of a 14mer oligonucleotide with a 5 bp duplex stem structure had previously been shown to bind to RTA with a K(d) of 480 nM, which improved to 12 nM upon addition of adenine. Second-generation stem-tetraloop inhibitors have been synthesized that incorporate a methylene bridge between the nitrogen of a 1-azasugar mimic, namely, (3S,4R)-3-hydroxy-4-(hydroxymethyl)pyrrolidine, and substituents, including phenyl, 8-aza-9-deazaadenyl, and 9-deazaadenyl groups, that mimic the activated leaving group at the transition state. The values for the dissociation constants (K(i)) for these were 99 nM for the phenyl 10mer, 163 and 94 nM for the 8-aza-9-deazaadenyl 10- and 14mers, respectively, and 280 nM for the 9-deazaadenyl 14mer. All of these compounds are among the tightest binding molecules known for RTA. A related phenyl-substituted inhibitor with a deoxyguanosine on the 5'-side of the depurination site was also synthesized on the basis of stem-loop substrate specificity studies. This molecule binds with a K(i) of 26 nM and is the tightest binding "one-piece" inhibitor. 8-Aza-9-deaza- and 9-deazaadenyl substituents provide an increased pK(a) at N7, a protonation site en route to the transition state. The binding of these inhibitors is not improved relative to the binding of their phenyl counterpart, however, suggesting that RTA might also employ protonation at N1 and N3 of the adenine moiety to activate the substrate during catalysis. Studies with methylated adenines support this argument. That the various stem-loop inhibitors have similar potencies suggests that an optimal one-piece inhibitor remains to be identified. The second-generation inhibitors described here incorporate ribose mimics missing the 2-hydroxy group. On the basis of inhibition data and substrate specificity studies, the 2'-hydroxyl group at the depurination site seems to be critical for recruitment as well as catalysis by RTA.     

Cutting edge: TGF-beta signaling is required for the in vivo expansion and immunosuppressive capacity of regulatory CD4+CD25+ T cells

Data regarding the role of TGF-beta for the in vivo function of regulatory CD4(+)CD25(+) T cells (Treg) are controversial. A transgenic mouse model with impaired TGF-beta signaling specifically in T cells was used to assess the role of endogenous TGF-beta for the in vivo function of CD4(+)CD25(+) Treg in a murine model of colitis induced by dextran sulfate. Transfer of wild-type, but not transgenic CD4(+)CD25(+) Treg was found to suppress colitis in wild-type mice. In addition, by transferring CFSE-labeled CD4(+)CD25(+) Treg we could demonstrate that endogenous TGF-beta promotes the expansion of CD4(+)CD25(+) Treg in vivo. Transgenic mice themselves developed reduced numbers of peripheral CD4(+)CD25(+) Treg and were more susceptible to the induction of colitis, which could be prevented by the transfer of wild-type Treg. These data indicate that TGF-beta signaling in CD4(+)CD25(+) Treg is required for their in vivo expansion and suppressive capacity.     

Prions adhere to soil minerals and remain infectious

An unidentified environmental reservoir of infectivity contributes to the natural transmission of prion diseases (transmissible spongiform encephalopathies [TSEs]) in sheep, deer, and elk. Prion infectivity may enter soil environments via shedding from diseased animals and decomposition of infected carcasses. Burial of TSE-infected cattle, sheep, and deer as a means of disposal has resulted in unintentional introduction of prions into subsurface environments. We examined the potential for soil to serve as a TSE reservoir by studying the interaction of the disease-associated prion protein (PrP(Sc)) with common soil minerals. In this study, we demonstrated substantial PrP(Sc) adsorption to two clay minerals, quartz, and four whole soil samples. We quantified the PrP(Sc)-binding capacities of each mineral. Furthermore, we observed that PrP(Sc) desorbed from montmorillonite clay was cleaved at an N-terminal site and the interaction between PrP(Sc) and Mte was strong, making desorption of the protein difficult. Despite cleavage and avid binding, PrP(Sc) bound to Mte remained infectious. Results from our study suggest that PrP(Sc) released into soil environments may be preserved in a bioavailable form, perpetuating prion disease epizootics and exposing other species to the infectious agent.     

Homer Wheelon, M.D., physiologist, artist, and poet: origins of the tailpieces in journals of the American Physiological Society

Since 1953, illustrations have been inserted as "tailpieces" at the ends of articles in The American Journal of Physiology and The Journal of Applied Physiology. The drawings were made by Homer Wheelon, a member of the American Physiological Society from 1919 until his death in 1960. Forty-five years after his death, Wheelon is unknown, but he contributed 32 publications to the medical literature and trained J. Earl Thomas, an important 20th century gastrointestinal physiologist. Wheelon was born into poverty in 1883 to itinerant Methodist preachers, circumstances that guided his education and career choices. Throughout his life, Wheelon exhibited a fondness and talent for art and photography and an unusual breadth of intellectual interests and knowledge. Wheelon received a bachelor's degree from the University of Washington, then studied at the University of Oregon, Northwestern University, and St. Louis University. Earning his M.D. from St. Louis University and assuming a faculty position there, Wheelon and his graduate student, Thomas, conducted widely recognized gastrointestinal research. Returning to Seattle in 1921, Wheelon became a highly respected physician and hospital administrator, but he also found time to indulge his interest in visual art and poetry. In 1933, inspired by observing a rabbit being used in a pregnancy test, Wheelon began to write and illustrate an epic, 322-page poem, Rabbit No. 202, illustrations from which became the journals' tailpieces. The present study traces Wheelon's personal life and scientific career in an attempt to understand this complex man and the origins of his unusual poem and its drawings.     

The reaction of phosphohexomutase from Pseudomonas aeruginosa: structural insights into a simple processive enzyme

The enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) from Pseudomonas aeruginosa catalyzes the reversible conversion of 1-phospho to 6-phospho-sugars. The reaction entails two phosphoryl transfers, with an intervening 180 degrees reorientation of the reaction intermediate (e.g. glucose 1,6-bisphosphate) during catalysis. Reorientation of the intermediate occurs without dissociation from the active site of the enzyme and is, thus, a simple example of processivity, as defined by multiple rounds of catalysis without release of substrate. Structural characterization of two PMM/PGM-intermediate complexes with glucose 1,6-bisphosphate provides new insights into the reaction catalyzed by the enzyme, including the reorientation of the intermediate. Kinetic analyses of site-directed mutants prompted by the structural studies reveal active site residues critical for maintaining association with glucose 1,6-bisphosphate during its unique dynamic reorientation in the active site of PMM/PGM.     

Interleukin-10 does not mediate inhalational tolerance in a chronic model of ovalbumin-induced allergic airway disease

OBJECTIVE: IL-10 is a potent anti-inflammatory cytokine, and IL-10-producing regulatory T cells are effective inhibitors of murine asthmatic responses. This study determined whether IL-10-dependent mechanisms mediated the local inhalational tolerance seen with chronic inhalational exposure to antigen. METHODS: Wildtype and IL-10(-/-) mice were sensitized with ovalbumin (OVA) and then challenged with daily OVA inhalations for 10 days or 6 weeks. RESULTS: The 10-day animals developed allergic airway disease, characterized by BAL eosinophilia, histologic airway inflammation and mucus secretion, methacholine hyperresponsiveness, and OVA-specific IgE production. These changes were more pronounced in IL-10(-/-) mice. The 6-week IL-10(-/-) and wildtype animals both developed inhalational tolerance, with resolution of airway inflammation but persistence of OVA-specific IgE production. CONCLUSION: IL-10 may have anti-inflammatory effects in the acute stage of murine allergic airways disease, but the cytokine does not mediate the development of local inhalational tolerance with chronic antigen exposure.