5 research outputs found
The Effects of A21G Mutation on Transmembrane Amyloid Beta (11â40) Trimer: An <i>In Silico</i> Study
Familial
Alzheimerâs disease (FAD) is passed down in family,
which account for 2â3% of about 40 million AD cases worldwide.
The Flemish (A21G) mutant of amyloid β (Aβ) exhibits unique
properties among all hereditary mutants of FAD, including the lowest
aggregation rate. Recent studies showed that Aβ oligomers play
a key role in Alzheimerâs disease (AD) pathogenesis. They could
insert themselves in brain cell membrane, disrupting the membrane
integrity and ion homeostasis. However, experimental studies of transmembrane
Aβ oligomers have been limited due to their intrinsic heterogeneity.
In this work, we extensively studied the A21G mutant of the transmembrane
3Aβ<sub>11â40</sub> (A21G 3Aβ<sub>11â40</sub>) using temperature replica exchange molecular dynamics (REMD) simulations.
Results provide detailed information on the conformational distribution
and dynamics of transmembrane A21G 3Aβ<sub>11â40</sub>. Minimal local change from A to G leads to significant conformational
changes and wider free energy holes on the free energy surface as
well as altered surface charges that lead to weaker affinity to the
dipalmitoylphosphatidylcholine (DPPC) lipid bilayers. These results
are consistent with experimental data that showed that A21G mutants
of Aβ peptides have lower aggregation rates and membrane binding
rates
Determinants of Regioselective Hydroxylation in the Fungal Polysaccharide Monooxygenases
The ubiquitous fungal polyÂsaccharide
monoÂoxyÂgenases
(PMOs) (also known as GH61 proteins, LPMOs, and AA9 proteins) are
structurally related but have significant variation in sequence. A
heterologous expression method in <i>Neurospora crassa</i> was developed as a step toward connecting regioselectivity of the
chemistry to PMO phylogeny. Activity assays, as well as sequence and
phylogenetic analyses, showed that the majority of fungal PMOs fall
into three major groups with distinctive active site surface features.
PMO1s and PMO2s hydroxylate glycosidic positions C1 and C4, respectively.
PMO3s hydroxylate both C1 and C4. A subgroup of PMO3s (PMO3*) hydroxylate
C1. Mutagenesis studies showed that an extra subdomain of about 12
amino acids contribute to C4 oxidation in the PMO3 family
An Unusual Peroxo Intermediate of the Arylamine Oxygenase of the Chloramphenicol Biosynthetic Pathway
Streptomyces venezuelae CmlI catalyzes
the six-electron oxygenation of the arylamine precursor of chloramphenicol
in a nonribosomal peptide synthetase (NRPS)-based pathway to yield
the nitroaryl group of the antibiotic. Optical, EPR, and MoĚssbauer
studies show that the enzyme contains a nonheme dinuclear iron cluster.
Addition of O<sub>2</sub> to the diferrous state of the cluster results
in an exceptionally long-lived intermediate (<i>t</i><sub>1/2</sub> = 3 h at 4 °C) that is assigned as a peroxodiferric
species (CmlI-peroxo) based upon the observation of an <sup>18</sup>O<sub>2</sub>-sensitive resonance Raman (rR) vibration. CmlI-peroxo
is spectroscopically distinct from the well characterized and commonly
observed <i>cis</i>-Ο-1,2-peroxo (Ο-Ρ<sup>1</sup>:Ρ<sup>1</sup>) intermediates of nonheme diiron enzymes.
Specifically, it exhibits a blue-shifted broad absorption band around
500 nm and a rR spectrum with a νÂ(OâO) that is at least
60 cm<sup>â1</sup> lower in energy. MoĚssbauer studies
of the peroxo state reveal a diferric cluster having iron sites with
small quadrupole splittings and distinct isomer shifts (0.54 and 0.62
mm/s). Taken together, the spectroscopic comparisons clearly indicate
that CmlI-peroxo does not have a Ο-Ρ<sup>1</sup>:Ρ<sup>1</sup>-peroxo ligand; we propose that a Ο-Ρ<sup>1</sup>:Ρ<sup>2</sup>-peroxo ligand accounts for its distinct spectroscopic
properties. CmlI-peroxo reacts with a range of arylamine substrates
by an apparent second-order process, indicating that CmlI-peroxo is
the reactive species of the catalytic cycle. Efficient production
of chloramphenicol from the free arylamine precursor suggests that
CmlI catalyzes the ultimate step in the biosynthetic pathway and that
the precursor is not bound to the NRPS during this step
Distal Hydrophobic Loop Modulates the Copper Active Site and Reaction of AA13 Polysaccharide Monooxygenases
Polysaccharide
monooxygenases (PMOs) use a type-2 copper
center
to activate O2 for the selective hydroxylation of one of
the two CâH bonds of glycosidic linkages. Our electron paramagnetic
resonance (EPR) analysis and molecular dynamics (MD) simulations suggest
the unprecedented dynamic roles of the loop containing the residue
G89 (G89 loop) on the active site structure and reaction cycle of
starch-active PMOs (AA13 PMOs). In the Cu(II) state, the G89 loop
could switch between an âopenâ and âclosedâ
conformation, which is associated with the binding and dissociation
of an aqueous ligand in the distal site, respectively. The conformation
of the G89 loop influences the positioning of the copper center on
the preferred substrate of AA13 PMOs. The dissociation of the distal
ligand results in the bending of the T-shaped core of the Cu(II) active
site, which could help facilitate its reduction to the active Cu(I)
state. In the Cu(I) state, the G89 loop is in the âclosedâ
conformation with a confined copper center, which could allow for
efficient O2 binding. In addition, the G89 loop remains
in the âclosedâ conformation in the Cu(II)-superoxo
intermediate, which could prevent off-pathway superoxide release via
exchange with the distal aqueous ligand. Finally, at the end of the
reaction cycle, aqueous ligand binding to the distal site could switch
the G89 loop to the âopenâ conformation and facilitate
product release
Distal Hydrophobic Loop Modulates the Copper Active Site and Reaction of AA13 Polysaccharide Monooxygenases
Polysaccharide
monooxygenases (PMOs) use a type-2 copper
center
to activate O2 for the selective hydroxylation of one of
the two CâH bonds of glycosidic linkages. Our electron paramagnetic
resonance (EPR) analysis and molecular dynamics (MD) simulations suggest
the unprecedented dynamic roles of the loop containing the residue
G89 (G89 loop) on the active site structure and reaction cycle of
starch-active PMOs (AA13 PMOs). In the Cu(II) state, the G89 loop
could switch between an âopenâ and âclosedâ
conformation, which is associated with the binding and dissociation
of an aqueous ligand in the distal site, respectively. The conformation
of the G89 loop influences the positioning of the copper center on
the preferred substrate of AA13 PMOs. The dissociation of the distal
ligand results in the bending of the T-shaped core of the Cu(II) active
site, which could help facilitate its reduction to the active Cu(I)
state. In the Cu(I) state, the G89 loop is in the âclosedâ
conformation with a confined copper center, which could allow for
efficient O2 binding. In addition, the G89 loop remains
in the âclosedâ conformation in the Cu(II)-superoxo
intermediate, which could prevent off-pathway superoxide release via
exchange with the distal aqueous ligand. Finally, at the end of the
reaction cycle, aqueous ligand binding to the distal site could switch
the G89 loop to the âopenâ conformation and facilitate
product release