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Clostridium difficile causes life-threatening diarrhea and is the leading cause of healthcare-associated bacterial infections in the United States. TcdA and TcdB bacterial toxins are primary determinants of disease pathogenesis and are attractive therapeutic targets. TcdA and TcdB contain domains that use UDP-glucose to glucosylate and inactivate host Rho GTPases, resulting in cytoskeletal changes causing cell rounding and loss of intestinal integrity. Transition state analysis revealed glucocationic character for the TcdA and TcdB transition states. We identified transition state analogue inhibitors and characterized them by kinetic, thermodynamic and structural analysis. Iminosugars, isofagomine and noeuromycin mimic the transition state and inhibit both TcdA and TcdB by forming ternary complexes with Tcd and UDP, a product of the TcdA- and TcdB-catalyzed reactions. Both iminosugars prevent TcdA- and TcdB-induced cytotoxicity in cultured mammalian cells by preventing glucosylation of Rho GTPases. Iminosugar transition state analogues of the Tcd toxins show potential as therapeutics for C. difficile pathology.

Heavy-enzyme isotope effects ( 15 N-, 13 C-, and 2 H-labeled protein) explore mass-dependent vibrational modes linked to catalysis. Transition path-sampling (TPS) calculations have predicted femtosecond dynamic coupling at the catalytic site of human purine nucleoside phosphorylase (PNP). Coupling is observed in heavy PNPs, where slowed barrier crossing caused a normal heavyenzyme isotope effect (k chem light /k chem heavy > 1.0). We used TPS to design mutant F159Y PNP, predicted to improve barrier crossing for heavy F159Y PNP, an attempt to generate a rare inverse heavyenzyme isotope effect (k chem light /k chem heavy < 1.0). Steady-state kinetic comparison of light and heavy native PNPs to light and heavy F159Y PNPs revealed similar kinetic properties. Pre-steadystate chemistry was slowed 32-fold in F159Y PNP. Pre-steady-state chemistry compared heavy and light native and F159Y PNPs and found a normal heavy-enzyme isotope effect of 1.31 for native PNP and an inverse effect of 0.75 for F159Y PNP. Increased isotopic mass in F159Y PNP causes more efficient transition state formation. Independent validation of the inverse isotope effect for heavy F159Y PNP came from commitment to catalysis experiments. Most heavy enzymes demonstrate normal heavy-enzyme isotope effects, and F159Y PNP is a rare example of an inverse effect. Crystal structures and TPS dynamics of native and F159Y PNPs explore the catalytic-site geometry associated with these catalytic changes. Experimental validation of TPS predictions for barrier crossing establishes the connection of rapid protein dynamics and vibrational coupling to enzymatic transition state passage.heavy enzyme | transition path sampling | purine nucleoside phosphorylase | enzyme design | femtosecond dynamics D ynamic motions essential for enzyme catalysis occur on timescales from milliseconds for conformational changes to femtosecond bond vibrations associated with chemistry at catalytic sites. The millisecond motions are linked to structural changes during substrate binding (1) and product release, whereas the femtosecond motions are involved in transition state (TS) formation. Alterations of the femtosecond dynamics by isotope substitution in enzymes influence the probability of TS barrier crossing when protein femtosecond motions are coupled to chemistry at catalytic sites (2, 3).The femtosecond dynamical effects in heavy purine nucleoside phosphorylase (PNP) on catalysis have been observed experimentally and have been explained by computational transition path sampling (TPS) (4, 5). TPS can provide insight into the atomic details of chemical reactions without prior knowledge of the reaction coordinate (6-8).Human PNP is a homotrimer that catalyzes the reversible phosphorolysis of 6-oxypurine nucleosides and 6-oxypurine-2′-deoxynucleosides to generate the corresponding purine bases and α-D-ribose (or 2-deoxy-α-D-ribose) 1-phosphates (Fig.

causes the most lethal form of human malaria and is a global health concern. The parasite responds to antimalarial therapies by developing drug resistance. The continuous development of new antimalarials with novel mechanisms of action is a priority for drug combination therapies. The use of transition-state analog inhibitors to block essential steps in purine salvage has been proposed as a new antimalarial approach. Mutations that reduce transition-state analog binding are also expected to reduce the essential catalytic function of the target. We have previously reported that inhibition of host and purine nucleoside phosphorylase (PNP) by DADMe-Immucillin-G (DADMe-ImmG) causes purine starvation and parasite death in vitro and in primate infection models. cultured under incremental DADMe-ImmG drug pressure initially exhibited increasedPNP gene copy number and protein expression. At increased drug pressure, additional PNP gene copies appeared with point mutations at catalytic site residues involved in drug binding. MutantPNPs from resistant clones demonstrated reduced affinity for DADMe-ImmG, but also reduced catalytic efficiency. The catalytic defects were partially overcome by gene amplification in the region expressing PNP. Crystal structures of native and mutatedPNPs demonstrate altered catalytic site contacts to DADMe-ImmG. Both point mutations and gene amplification are required to overcome purine starvation induced by DADMe-ImmG. Resistance developed slowly, over 136 generations (2 clonal selection). Transition-state analog inhibitors against PNP are slow to induce resistance and may have promise in malaria therapy.

Crystal structures of human mitochondrial 3-ketoacyl-CoA thiolase (hT1) in the apo form and in complex with CoA have been determined at 2.0 Å resolution. The structures confirm the tetrameric quaternary structure of this degradative thiolase. The active site is surprisingly similar to the active site of the Zoogloea ramigera biosynthetic tetrameric thiolase (PDB entries 1dm3 and 1m1o) and different from the active site of the peroxisomal dimeric degradative thiolase (PDB entries 1afw and 2iik). A cavity analysis suggests a mode of binding for the fatty-acyl tail in a tunnel lined by the Nβ2-Nα2 loop of the adjacent subunit and the Lα1 helix of the loop domain. Soaking of the apo hT1 crystals with octanoyl-CoA resulted in a crystal structure in complex with CoA owing to the intrinsic acyl-CoA thioesterase activity of hT1. Solution studies confirm that hT1 has low acyl-CoA thioesterase activity for fatty acyl-CoA substrates. The fastest rate is observed for the hydrolysis of butyryl-CoA. It is also shown that T1 has significant biosynthetic thiolase activity, which is predicted to be of physiological importance.