3 research outputs found
Conformational Analysis and Parallel QM/MM X‑ray Refinement of Protein Bound Anti-Alzheimer Drug Donepezil
The
recognition and association of donepezil with acetylcholinesterase
(AChE) has been extensively studied in the past several decades because
of the former’s use as a palliative treatment for mild Alzheimer
disease. Herein, we examine the conformational properties of donepezil
and we re-examine the donepezil-AChE crystal structure using combined
quantum mechanical/molecular mechanical (QM/MM) X-ray refinement tools.
Donepezil’s conformational energy surface was explored using
the M06 suite of density functionals and with the MP2/complete basis
set (CBS) method using the aug-cc-pVXZ (<i>X</i> = D and
T) basis sets. The donepezil-AChE complex (PDB 1EVE) was
also rerefined through a parallel QM/MM X-ray refinement approach
based on an in-house ab initio code QUICK, which uses
the message passing interface (MPI) in a distributed SCF algorithm
to accelerate the calculation via parallelization. In the QM/MM rerefined
donepezil structure, coordinate errors that previously existed in
the PDB deposited geometry were improved leading to an improvement
of the modeling of the interaction between donepezil and the aromatic
side chains located in the AChE active site gorge. As a result of
the rerefinement there was a 93% reduction in the donepezil conformational
strain
energy versus the original PDB structure. The results of the present
effort offer further detailed structural and biochemical inhibitor-AChE
information for the continued development of more effective and palliative
treatments of Alzheimer disease
Catalytic Mechanism of Aromatic Prenylation by NphB
NphB is an aromatic prenyltransferase that catalyzes
the attachment
of a 10-carbon geranyl group to aromatic substrates. Importantly,
NphB exhibits a rich substrate selectivity and product regioselectivity.
A systematic computational study has been conducted in order to address
several question associated with NphB-catalyzed geranylation. The
reaction mechanism of the prenylation step has been characterized
as a S<sub>N</sub>1 type dissociative mechanism with a weakly stable
carbocation intermediate. A novel π-chamber composed of Tyr121,
Tyr216, and 1,6-DHN is found to be important in stabilizing the carbocation.
The observed difference in the rates of product formation from 5-
and 2-prenylation arises from the differing orientations of the aromatic
substrate in the resting state. 4-Prenylation shares the same resting
state with 5-prenylation, but the lower free energy barrier for carbocation
formation makes the latter reaction more facile. The high free energy
barrier associated with 7-prenylation is caused by the unfavorable
orientation of 1,6-DHN in active site pocket, along with the difficulty
of proton elimination after the prenylation step. A water-mediated
proton transfer facilitates the loss of hydrogen at the prenylation
site to form the final prenylated product. Interestingly, the same
crystallographically observed water molecule has been found to be
responsible for proton loss in all three experimentally identified
products. After proton transfer, the relaxation of the final product
from a sp<sup>3</sup> carbon center to a sp<sup>2</sup> center triggers
a “spring-loaded” product release mechanism which pushes
the final product out of the binding pocket toward the edge of the
active site. The hydrogen bond interactions between the two hydroxyl
groups of the aromatic product and the side chains of Ser214 and Tyr288
help to “steer” the movement of the product. In addition,
mutagenesis studies identify these same two side chains as being responsible
for the observed regioselectivity, particularly 2-prenylation. These
observations provide valuable insights into NphB chemistry, offering
an opportunity to better engineer the active site and to control the
reactivity in order to obtain high yields of the desired product(s).
Furthermore, the S<sub>N</sub>1 reaction mechanism observed for NphB
differs from the prenylation reaction found in, for example, the farnesyltransferase,
which proceeds via an S<sub>N</sub>2-like reaction pathway. The spring-loaded
release mechanism highlighted herein also offers novel insights into
how enzymes facilitate product release
Mechanism of Formation of the Nonstandard Product in the Prenyltransferase Reaction of the G115T Mutant of FtmPT1: A Case of Reaction Dynamics Calling the Shots?
FtmPT1
is a fungal indole prenyltransferase that affords Tryprostatin
B from Brevianamide F and dimethylallyl pyrophosphate; however, when
a single residue in the active site is mutated (Gly115Thr), a novel
five-membered ring compound is obtained as the major product with
Tryprostatin B as the minor product. Herein, we describe detailed
studies of the catalysis of the Gly115Thr mutant of FtmPT1 with a
focus on the observed regioselectivity of the reaction. We employ
one- and two-dimensional potential of mean force simulations to explore
the catalytic mechanism, along with molecular dynamics simulations
exploring the reaction dynamics of the prenyl transfer reaction. Single-point
electronic structure calculations were also used to explore the performance
of the self-consistent charge density functional tight-binding method
to model specific reaction steps. Importantly, we observe that the
two reaction pathways have comparable activation parameters and propose
that the origin of the novel product is predicated, at least in part,
on the topology of the potential energy surface as revealed by reaction
dynamics studies