Development of disease-modifying therapeutics is urgently needed for treating Alzheimer disease (AD). AD is characterized by toxic β-amyloid (Aβ) peptides produced by β- and γ-secretase-mediated cleavage of the amyloid precursor protein (APP). β-secretase inhibitors reduce Aβ levels, but mechanism-based side effects arise because they also inhibit β-cleavage of non-amyloid substrates like Neuregulin. We report that β-secretase has a higher affinity for Neuregulin than it does for APP. Kinetic studies demonstrate that the affinities and catalytic efficiencies of β-secretase are higher toward non-amyloid substrates than toward APP. We show that non-amyloid substrates are processed by β-secretase in an endocytosis-independent manner. Exploiting this compartmentalization of substrates, we specifically target the endosomal β-secretase by an endosomally targeted β-secretase inhibitor, which blocked cleavage of APP but not non-amyloid substrates in many cell systems, including induced pluripotent stem cell (iPSC)-derived neurons. β-secretase inhibitors can be designed to specifically inhibit the Alzheimer process, enhancing their potential as AD therapeutics without undesired side effects.

NO dioxygenation, i.e., the oxidation of nitric oxide to nitrate by oxygen-bound truncated hemoglobin (trHbN) is studied using reactive molecular dynamics simulations. This reaction is an important step in a sequence of events in the overall NO detoxification reaction involving trHbN. The simulations ( approximately 160 ns in total) reveal that the reaction favors a pathway including (i) NO binding to oxy-trHbN, followed by (ii) rearrangement of peroxynitrite-trHbN to nitrato-trHbN, and finally (iii) nitrate dissociation from nitrato-trHbN. Overall, the reactions occur within tens of picoseconds and the crossing seam of the reactant and product are found to be broad. The more conventional pathway, where the peroxynitrite-trHbN complex undergoes peroxide cleavage to form free NO(2) and oxo-ferryl trHbN, is found to be too slow due to a considerable barrier involved in peroxide bond dissociation. The energetics of this step is consistent with earlier electronic structure calculations and make this pathway less likely. The role of Tyr33 and Gln58 in the NO dioxygenation has been investigated by studying the reaction in mutants of trHbN. The mutation study suggests that residues Tyr33 and Gln58 preorient the reactive ligands through a highly dynamical H-bonding network which facilitates the reaction. In particular, the Y33A mutation leads to a significant retardation in NO dioxygenation, in agreement with experiments which reveal a strong influence of the protein environment on the reaction rate.

Atomistic simulations of nitric oxide (NO) dynamics and migration in the trHbN of Mycobacterium tuberculosis are reported. From extensive molecular dynamics simulations (48 ns in total), the structural and energetic properties of the ligand docking sites in the protein have been characterized and a connectivity network between the ligand docking sites has been built. Several novel migration and exit pathways are found and are analyzed in detail. The interplay between a hydrogen-bonding network involving residues Tyr(33) and Gln(58) and the bound O(2) ligand is discussed and the role of Phe(62) residue in ligand migration is examined. It is found that Phe(62) is directly involved in controlling ligand migration. This is reminiscent of His(64) in myoglobin, which also plays a central role in CO migration pathways. Finally, infrared spectra of the NO molecule in different ligand docking sites of the protein are calculated. The pocket-specific spectra are typically blue-shifted by 5-10 cm(-1), which should be detectable in future spectroscopic experiments.

With increasing cases of fatal bacterial infections and growing antibiotic resistance, unrelenting efforts are necessary for identification of novel antibiotic targets and new drug molecules. The dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) is a di-nuclear Zn containing enzyme in the lysine biosynthetic pathway which is indispensable for bacterial survival and absent in the human host, thus a potential antibiotic target. The DapE enzyme catalyzes the hydrolysis of N-succinyl-L,L-diaminopimelic acid (SDAP) to give rise to succinic acid and L,L-diaminopimelic acid. The mechanism of action of the DapE catalyzed SDAP hydrolysis is investigated employing a hybrid QM/MM computational method. The DapE side chains, such as, Arg178, Thr325, Asn345, are found to play a role in substrate identification and stabilization of the enzyme active site. Furthermore, a glycine rich loop (Gly322-Ser326) is found to facilitate tight binding of the substrate in the enzyme active site. The catalytic reaction progresses via a general acid-base hydrolysis mechanism where Glu134 first acts as a Lewis base by activating the catalytic water molecule in the active site, followed by guiding the resulting hydroxyl ion for a nucleophilic attack on the substrate, and finally acts as a Lewis acid by donating a proton to the substrate. The intermediates and transition states along the reaction pathway have been structurally and energetically characterized. A conformational change in the side chain of Asp100, which bridges the two Zn centers of the enzyme, is observed which facilitates the enzymatic action by lowering the activation energy and leads to the formation of a new intermediate during the catalytic reaction. The nucleophilic attack is found to be the rate determining step.

Mononuclear Fe(iii) complexes commonly exist in high-spin or low-spin states, whereas their occurrence in the intermediate-spin state (S = 3/2) is scarce. The magnetic anisotropy in two trigonal-bipyramidal mononuclear Fe(iii) complexes, (PMe3)2FeCl3 (1) and (PMe2Ph)2FeCl3 (2), in their intermediate-spin ground state has been examined by ab initio electronic structure calculations. The calculations successfully reproduce the experimental magnetic anisotropic barrier, Ueff in 1 (81 cm−1) and 2 (42 cm−1), which is shown to arise due to thermally assisted quantum tunneling of magnetization from the second Kramer’s doublets. The magnetic anisotropy in both the complexes is found to be significantly influenced by the axial ligands, while the equatorial ligands have negligible contribution. The large reduction in Ueff of 2 has been shown to arise due to the phenyl groups, which results in the lifting of orbital degeneracy of e″ and e′ frontier orbitals and leads to a net quenching of the orbital angular momentum of the metal center causing a diminished spin-orbit splitting in 2. While the crystal structure of 2 shows two phenyl rings out of plane to each other, the present study discovered another stable conformation of 2, where the two phenyl rings are in the same plane (2a). Unlike 2, the planarity of the two phenyl rings in 2a restores the degeneracy of the frontier orbitals, thereby increasing the spin-orbit splitting and a consequent rise in Ueff from 42 to 80 cm−1 in 2a.