Elusive glycosyl-enzyme adduct: Using classical MD simulations and QM/MM metadynamics, the long-time sought glycosyl-enzyme covalent intermediate of a retaining glycosyltransferase, with a putative nucleophile residue in the active site, has been trapped (MD=molecular dynamics; QM/MM=quantum mechanics/molecular mechanics).
The catalytic mechanism of retaining glycosyltransferases (ret-GTs) remains a controversial issue in glycobiology. By analogy to the well-established mechanism of retaining glycosidases, it was first suggested that ret-GTs follow a double-displacement mechanism. However, only family 6 GTs exhibit a putative nucleophile protein residue properly located in the active site to participate in catalysis, prompting some authors to suggest an unusual single-displacement mechanism [named as front-face or SNi (substitution nucleophilic internal)-like]. This mechanism has now received strong support, from both experiment and theory, for several GT families except family 6, for which a double-displacement reaction is predicted. In the last few years, we have uncovered the molecular mechanisms of several retaining GTs by means of quantum mechanics/molecular mechanics (QM/MM) metadynamics simulations, which we overview in the present work.
DNA oligomers can form silver-mediated duplexes, stable in gas phase and solution, with potential for novel biomedical and technological applications. The nucleobase-metal bond primarily drives duplex formation, but hydrogen (H-) bonds may also be important for structure selection and stability. To elucidate the role of H-bonding, we conducted theoretical and experimental studies of a duplex formed by silver-mediated cytosine homopobase DNA strands, two bases long. This silver-mediated cytosine tetramer is small enough to permit accurate, realistic modeling by DFT-based quantum mechanics/molecular mechanics methods. In gas phase, our calculations found two energetically favorable configurations distinguished by H-bonding, one with a novel interplane H-bond, and the other with planar H-bonding of silver-bridged bases. Adding solvent favored silver-mediated tetramers with interplane H-bonding. Overall agreement of electronic circular dichroism spectra for the final calculated structure and experiment validates these findings. Our results can guide use of these stabilization mechanisms for devising novel metal-mediated DNA structures.
The effects of aqueous solvent and biological ligands on the structural and electronic properties of thiolate-protected Au25(SR)18(-) clusters have been studied by performing quantum mechanics/molecular mechanics (QM/MM) simulations. Analysis of bond distances and angles show that the solvated nanocluster experiences modest structural changes, which are reflected as flexibility of the Au core. The hydrophilic glutathione ligands shield the metallic core effectively and distort its symmetry via sterical hindrance effects. We show that the previously reported agreement between the calculated HOMO-LUMO gap of the cluster and the optical measurement is due to cancellation of errors, where the typical underestimation of the theoretical band gap compensates the effect of the missing solvent. The use of a hybrid functional results in a HOMO-LUMO gap value of 1.5 eV for the solvated nanocluster with glutathione ligands, in good agreement with optical measurements. Our results demonstrate that ligand/solvent effects should be considered for a proper comparison between theory and experiment.
Retaining glycoside hydrolases cleave their substrates through stereochemical retention at the anomeric position. Typically, this involves two-step mechanisms using either an enzymatic nucleophile via a covalent glycosyl enzyme intermediate or neighboring-group participation by a substrate-borne 2-acetamido neighboring group via an oxazoline intermediate; no enzymatic mechanism with participation of the sugar 2-hydroxyl has been reported. Here, we detail structural, computational, and kinetic evidence for neighboring-group participation by a mannose 2hydroxyl in glycoside hydrolase family 99 endo-α-1,2-mannanases. We present a series of crystallographic snapshots of key species along the reaction coordinate: a Michaelis complex with a tetrasaccharide substrate; complexes with intermediate mimics, a sugar-shaped cyclitol β-1,2-aziridine and β-1,2-epoxide; and a product complex. The 1,2-epoxide intermediate mimic displayed hydrolytic and transfer reactivity analogous to that expected for the 1,2-anhydro sugar intermediate supporting its catalytic equivalence. Quantum mechanics/molecular mechanics modeling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar via a transition state in an unusual flattened, envelope (E 3 ) conformation. Kinetic isotope effects (k cat /K M ) for anomeric-2 H and anomeric-13 C support an oxocarbenium ion-like transition state, and that for C2-18 O (1.052 ± 0.006) directly implicates nucleophilic participation by the C2-hydroxyl. Collectively, these data substantiate this unprecedented and long-imagined enzymatic mechanism.
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