Catalytic mechanism investigation of lysine-specific demethylase 1 (LSD1): a computational study.
ABSTRACT Lysine-specific demethylase 1 (LSD1), the first identified histone demethylase, is a flavin-dependent amine oxidase which specifically demethylates mono- or dimethylated H3K4 and H3K9 via a redox process. It participates in a broad spectrum of biological processes and is of high importance in cell proliferation, adipogenesis, spermatogenesis, chromosome segregation and embryonic development. To date, as a potential drug target for discovering anti-tumor drugs, the medical significance of LSD1 has been greatly appreciated. However, the catalytic mechanism for the rate-limiting reductive half-reaction in demethylation remains controversial. By employing a combined computational approach including molecular modeling, molecular dynamics (MD) simulations and quantum mechanics/molecular mechanics (QM/MM) calculations, the catalytic mechanism of dimethylated H3K4 demethylation by LSD1 was characterized in details. The three-dimensional (3D) model of the complex was composed of LSD1, CoREST, and histone substrate. A 30-ns MD simulation of the model highlights the pivotal role of the conserved Tyr761 and lysine-water-flavin motif in properly orienting flavin adenine dinucleotide (FAD) with respect to substrate. The synergy of the two factors effectively stabilizes the catalytic environment and facilitated the demethylation reaction. On the basis of the reasonable consistence between simulation results and available mutagenesis data, QM/MM strategy was further employed to probe the catalytic mechanism of the reductive half-reaction in demethylation. The characteristics of the demethylation pathway determined by the potential energy surface and charge distribution analysis indicates that this reaction belongs to the direct hydride transfer mechanism. Our study provides insights into the LSD1 mechanism of reductive half-reaction in demethylation and has important implications for the discovery of regulators against LSD1 enzymes.
- SourceAvailable from: stimes.cn[show abstract] [hide abstract]
ABSTRACT: An important development in understanding the influence of chromatin on gene regulation has been the finding that DNA methylation and histone post-translational modifications lead to the recruitment of protein complexes that regulate transcription. Early interpretations of this phenomenon involved gene regulation reflecting predictive activating or repressing types of modification. However, further exploration reveals that transcription occurs against a backdrop of mixtures of complex modifications, which probably have several roles. Although such modifications were initially thought to be a simple code, a more likely model is of a sophisticated, nuanced chromatin 'language' in which different combinations of basic building blocks yield dynamic functional outcomes.Nature 06/2007; 447(7143):407-12. · 38.60 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The surface of nucleosomes is studded with a multiplicity of modifications. At least eight different classes have been characterized to date and many different sites have been identified for each class. Operationally, modifications function either by disrupting chromatin contacts or by affecting the recruitment of nonhistone proteins to chromatin. Their presence on histones can dictate the higher-order chromatin structure in which DNA is packaged and can orchestrate the ordered recruitment of enzyme complexes to manipulate DNA. In this way, histone modifications have the potential to influence many fundamental biological processes, some of which may be epigenetically inherited.Cell 03/2007; 128(4):693-705. · 31.96 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Posttranslational modifications of histone N-terminal tails impact chromatin structure and gene transcription. While the extent of histone acetylation is determined by both acetyltransferases and deacetylases, it has been unclear whether histone methylation is also regulated by enzymes with opposing activities. Here, we provide evidence that LSD1 (KIAA0601), a nuclear homolog of amine oxidases, functions as a histone demethylase and transcriptional corepressor. LSD1 specifically demethylates histone H3 lysine 4, which is linked to active transcription. Lysine demethylation occurs via an oxidation reaction that generates formaldehyde. Importantly, RNAi inhibition of LSD1 causes an increase in H3 lysine 4 methylation and concomitant derepression of target genes, suggesting that LSD1 represses transcription via histone demethylation. The results thus identify a histone demethylase conserved from S. pombe to human and reveal dynamic regulation of histone methylation by both histone methylases and demethylases.Cell 01/2005; 119(7):941-53. · 31.96 Impact Factor
Catalytic Mechanism Investigation of Lysine-Specific
Demethylase 1 (LSD1): A Computational Study
Xiangqian Kong1., Sisheng Ouyang1., Zhongjie Liang1, Junyan Lu1, Liang Chen1, Bairong Shen2,
Donghai Li3, Mingyue Zheng1, Keqin Kathy Li4*, Cheng Luo1,2*, Hualiang Jiang1,5
1State Key Laboratory of Drug Research, Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China,
2Center for Systems Biology, Soochow University, Jiangsu, China, 3State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Jiangsu Diabetes
Research Center, Nanjing University, Nanjing, China, 4State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Rui Jin Hospital, Shanghai Jiao Tong
University School of Medicine, Shanghai, China, 5School of Pharmacy, East China University of Science and Technology, Shanghai, China
Lysine-specific demethylase 1 (LSD1), the first identified histone demethylase, is a flavin-dependent amine oxidase which
specifically demethylates mono- or dimethylated H3K4 and H3K9 via a redox process. It participates in a broad spectrum of
biological processes and is of high importance in cell proliferation, adipogenesis, spermatogenesis, chromosome
segregation and embryonic development. To date, as a potential drug target for discovering anti-tumor drugs, the medical
significance of LSD1 has been greatly appreciated. However, the catalytic mechanism for the rate-limiting reductive half-
reaction in demethylation remains controversial. By employing a combined computational approach including molecular
modeling, molecular dynamics (MD) simulations and quantum mechanics/molecular mechanics (QM/MM) calculations, the
catalytic mechanism of dimethylated H3K4 demethylation by LSD1 was characterized in details. The three-dimensional (3D)
model of the complex was composed of LSD1, CoREST, and histone substrate. A 30-ns MD simulation of the model
highlights the pivotal role of the conserved Tyr761 and lysine-water-flavin motif in properly orienting flavin adenine
dinucleotide (FAD) with respect to substrate. The synergy of the two factors effectively stabilizes the catalytic environment
and facilitated the demethylation reaction. On the basis of the reasonable consistence between simulation results and
available mutagenesis data, QM/MM strategy was further employed to probe the catalytic mechanism of the reductive half-
reaction in demethylation. The characteristics of the demethylation pathway determined by the potential energy surface
and charge distribution analysis indicates that this reaction belongs to the direct hydride transfer mechanism. Our study
provides insights into the LSD1 mechanism of reductive half-reaction in demethylation and has important implications for
the discovery of regulators against LSD1 enzymes.
Citation: Kong X, Ouyang S, Liang Z, Lu J, Chen L, et al. (2011) Catalytic Mechanism Investigation of Lysine-Specific Demethylase 1 (LSD1): A Computational
Study. PLoS ONE 6(9): e25444. doi:10.1371/journal.pone.0025444
Editor: Annalisa Pastore, National Institute for Medical Research, Medical Research Council, United Kingdom
Received June 14, 2011; Accepted September 5, 2011; Published September 30, 2011
Copyright: ? 2011 Kong et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors gratefully acknowledge financial support from the State Key Program of Basic Research of China grant (2009CB918502), the National
Natural Science Foundation of China grants (20972174, 21021063, 31000323 and 91029704), Shanghai Committee of Science and Technology grants
(10410703900), Specialized Research Fund for the Doctoral Program of Higher Education of China (20100091120023) and the Chinese Academy of Sciences
(XDA01040305). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org (CL); email@example.com (KKL)
. These authors contributed equally to this work.
Histones are subjected to a variety of post-translational
modifications, including acetylation, phosphorylation, ubiquitina-
tion, sumolation and methylation [1,2]. These modifications
steadily modulate chromatin structure and the consequent diverse
chromatin-based processes in a combined fashion. Distinguished
from other modifications that are dynamically regulated, histone
methylation has long been thought to be a permanent epigenetic
marker. However, the recent discovery of lysine-specific demethy-
lase 1 (LSD1) and Jumonji domain-containing proteins strongly
challenged this dogma, by demonstrating that histone lysine
methylation can be actively and dynamically regulated by histone
methylase and demethylase .
LSD1, the first identified histone demethylase, is a flavin-
dependent amine oxidase and conserved from Schizosaccharo-
myces pombe to mammals . As a member of monoamine
oxidase (MAO) family, LSD1 catalyzes the specific demethylation
of mono- or dimethylated histone H3 lysine4 (H3K4) and H3
lysine 9 (H3K9) via a redox process. As illustrated in Figure 1,
during the reductive half-reaction, the a-CH bond of the substrate
was oxidatively cleaved to form an imine intermediate with the
coinstantaneous transfer of a hydride equivalent to flavin-adenine
dinucleotide (FAD). The reduced cofactor was then reoxidated to
its functional form by molecular oxygen accompanied with the
release of hydrogen peroxide byproduct during the oxidative half-
reaction. The imine intermediate was further hydrolyzed non-
enzymatically to release the unmodified lysine and formaldehyde.
In addition to histone substrates, LSD1 can also act on non-
histone substrates, such as p53 [5,6], DNMT1 , and MYPT1
. Meanwhile, LSD1 was an integral component of miscella-
neous chromatin remodeling and transcriptional complexes
[9,10,11,12]. The ability to modulate such a wide range of
substrates and pathways has implied the pivotal role of LSD1 in a
PLoS ONE | www.plosone.org1 September 2011 | Volume 6 | Issue 9 | e25444
broad spectrum of biological processes, including cell proliferation
, adipogenesis , spermatogenesis , chromosome
segregation , pluripotency regulation of stem cell  and
embryonic development . The dysregulation of LSD1 activity
possesses a significant impact on human carcinogenesis  and has
been implicated in maintenance of a variety of cancer types, such
as neuroblastoma , breast cancer [20,21], colon cancer ,
etc. Moreover, the expression of LSD1 is closely correlated with
the relapse of prostate cancer during therapy [23,24].
However, for most of the flavin-dependent amine oxidases, a
major unraveled portion of the chemical mechanism is the
reductive half-reaction which involves the irreversible CH bond
cleavage. There are three major protracted controversies for this
issue which include hydride transfer mechanism , polar
nucleophilic mechanism  and radical mechanism . As
illustrated in Figure 2, hydride transfer mechanism may be the
most unequivocal process which only involves a direct transfer of a
hydride from the substrate a-carbon to flavin, while the polar
nucleophilic mechanism and radical mechanism necessitate an
adduct intermediate and radical intermediate preceding the CH
cleavage, respectively. Due to the significantly conserved archi-
tecture of the catalytic domain and the chemical nature of catalytic
reaction, it is conceivable that these debates are truly subsistent in
Taken together, the elaborate elucidation of catalytic mecha-
nism of LSD1 is not only of great fundamental interest, but also of
high medical importance since it would facilitate the development
of novel mechanism-based inactivators. Thus, in the present study,
the catalytic mechanism of LSD1 was investigated by combining
molecular modeling, MD simulations and QM/MM calculations.
The ternary complex structure of LSD1-CoREST-substrate in
aqueous solution was obtained from MD simulation. The
simulation results highlight that the conserved lysine–water–flavin
motif and Tyr761 may play a vital role in both properly orienting
FAD with respect to substrate and expediting the demethylation
process, which are consistent with the site-directed mutagenesis
results and related kinetic studies. In the end, the QM/MM
strategy was employed on the ternary complex structure, and the
results suggest that the reductive half-reaction of LSD1 undergoes
the hydride transfer process. These findings provide the atomic
description of the demethylation pathway and insights into the
molecular mechanism of LSD1.
Materials and Methods
Preparation of the simulation system
The initial configuration of the enzyme-substrate complex was
modeled on the basis of the crystal structure of LSD1 in complex
with CoREST, a co-repressor that enables LSD1 to demethylate
nucleosomal substrates (PDB code: 2IW5) , and LSD1-
CoREST-H3 peptide ternary complex (PDB code: 2V1D) .
First, the H3 peptide in ternary complex was extracted to the
LSD1-CoREST complex structure which has higher resolution
(R=2.57 A˚). Then, the H3 peptide was mutated to the valid
substrate structure of LSD1 by replacing the mutated methionine
residue with dimethylated lysine. The resulting LSD1-CoREST-
substrate complex was minimized by using the AMBER force field
with the following parameters: a distance-dependent dielectric
function, nonbonded cutoff of 8 A˚, Amber charges for the protein,
and Gastieger-Hu ¨ckel charges for dimethylated lysine and FAD.
The structure was minimized by the simplex method, followed by
the Powell method to an energy gradient ,0.05 kcal/(mol?A˚). All
procedures were performed using the Sybyl software package
(Tripos, St. Louis, MO).
Molecular dynamics simulation
MD simulations were performed on the LSD1-CoREST-
substrate complex structure. Before simulations, the protonation
states of ionizable residues were chosen based on the prediction of
H++ program  and carefully visual inspection of their local
electrostatic and hydrogen bond microenvironment. The complex
was solvated into a rectangular box with a 10 A˚buffer distance
between the solvent box wall and the nearest solute atoms. Then,
the complex-water system was subjected to energy minimization.
Figure 1. The proposed catalytic mechanism for the overall demethylation reaction of LSD1.
QM/MM Study of Catalytic Mechanism of LSD1
PLoS ONE | www.plosone.org2September 2011 | Volume 6 | Issue 9 | e25444
Afterward, counterions were added to the system to neutralize the
simulation system and the whole system was subsequently
minimized again. The charges of atoms of FAD and dimethylated
lysine were calculated by using the RESP method  encoded in
the AMBER suite of programs  at the level of RHF/6-31G*.
Covalent and nonbonded parameters for the atoms of FAD and
dimethylated lysine were assigned, by analogy or through
interpolation, from those already present in the AMBER force
All MD simulations were performed using the AMBER package
(version 10.0) with constant temperature and pressure (NPT) and
periodic boundary conditions. The Amber99SB [33,34,35] force
field for the protein and TIP3P model for water molecules
were employed. During MD simulations, all bonds involving
hydrogen atoms were constrained with the SHAKE algorithm
, and the integration step of 2 fs was used. Electrostatic
interactions were calculated using the particle-mesh Ewald method
. The nonbonded cutoff was set to 10.0 A˚, and the nonbonded
pairs were updated every 25 steps. The simulation was coupled to
a 300 K thermal bath at 1.0 atm of pressure (atm=101.3 kPa) by
applying the algorithm of Berendsen et al.  The temperature
and pressure coupling parameters were set as 1 ps.
QM/MM calculations were performed with the use of a two-
layered ONIOM scheme encoded in the Gaussian03 program
. The ONIOM method is a hybrid quantum chemical
approach developed by Morokuma and coworkers that allows
different levels of theory to be applied to different parts of a
molecular system [41,42,43,44,45,46,47]. In this approach, the
molecular system under investigation is defined as two parts. The
‘‘model’’ system consists of the most critical elements of the system
and is treated with an accurate (high-level) computational method
which can describe bond breaking and formation. The ‘‘real’’
system includes the entire system and is treated with an
inexpensive (low-level) computational method which can depict
the environmental effects of the molecular environment on the
‘‘model’’ system. The total ONIOM energy, EONIOM, is defined as
Where E(high, model) is the energy of the model system (includes
the link atoms) at the high level of theory, E(low, real) is the energy
of the real system at the low level of theory, and E(low, model) is
the energy of the model system at the low level of theory. Thus, the
ONIOM method allows one to perform a high-level calculation on
just a small, critical part of the molecular system and incorporate
the effects of the surrounding elements at a lower level of theory to
yield a consistent energy expression on the full system.
The quantum mechanical (QM) region consists of the
dimethylamino portion (-(CH2)3-N(CH3)2) of dimethylated H3K4
and the crucial parts of the conserved lysine-water-flavin motif,
which includes the flavin ring and its adjacent methylene group of
FAD, most of the side chain (-(CH2)3-NH2) of Lys661 and the
water molecular bridging the flavin and Lys661 through two
hydrogen bonds. Link hydrogen atoms [48,49] were employed to
saturate the dangling covalent bonds. The QM region comprises
66 atoms and was described in terms of the density functional
theorywith theB3LYP exchange-correlation
[50,51,52,53] (UB3LYP for open shell in the radical mechanism)
and 6-31G* basis set. The remainder of the system (MM region)
Figure 2. Three major suggested catalytic mechanism proposals for the reductive half-reaction of the flavin-dependent amine
QM/MM Study of Catalytic Mechanism of LSD1
PLoS ONE | www.plosone.org3 September 2011 | Volume 6 | Issue 9 | e25444
was treated by using the AMBER Parm99 force field. A total of
12984 atoms were included for the QM/MM calculations. The
electrostatic interactions between the QM and MM regions were
calculated by an electronic embedding scheme. The partial
charges of the MM region were incorporated into QM
Hamiltonian, which provides a better description of the electro-
static interaction between the QM and MM regions and allows the
QM wavefunction to be polarized. The charges for all the QM
atoms were fitted to the electrostatic potential at points selected
according to the Merz-Singh-Kollman scheme and calculated at
the B3LYP/6-31G* level. The minimized structure optimized
using the AMBER Parm99 force field  was further optimized
at the ONIOM (B3LYP/6-31G*:Amber) level.
Results and Discussion
The protonation states of key residues in active site
The acid dissociation constant (pKa) of ionizable residues was
determined computationally by H++ program  on the basis of
substrate-alone (histone substrate), substrate-free (LSD1-CoREST
complex, PDB code: 2IW5)  and substrate-bound (modeled
LSD1-CoREST-substrate complex, see Material and Methods)
states by using different internal dielectric constants. As shown in
Table 1, in the substrate-free state, the conserved Lys661 was
positive-charged with a normal pKa value, whereas binding of the
histone substrate within active site significantly reduced its pKa
values regardless of different internal dielectric constants, which
preserved Lys661 in neutral state under physiological environ-
ment. This difference can be attributed to the variation of the
microenvironment in active-site. The Lys661 is located at a
remarkably hydrophobic active-site channel underneath the
protein surface. The exact match of the substrate with the narrow
channel not only expelled solvent but also effectively sealed the
active center, which was a conserved feature among flavoprotein
amine oxidases [54,55,56]. Thus, such solvent inaccessibility of the
active center gave rise to strong electrostatic effect that alters the
acid/base equilibrium of Lys661. Similar results were obtained in
recent studies of the catalytic mechanism of maize polyamine
oxidase (MPAO)  and mammalian polyamine oxidase ,
two homologues of LSD1, which demonstrated the corresponding
conserved lysines in active center were deprotoned upon substrate
binding from both experimental and theoretical perspectives
[57,58,59]. Furthermore, resembling Lys661, the pKa value for
H3K4 in complex was remarkably lower than that of in bulk
solvent. In consideration of the same niche that H3K4 and Lys661
coexisted, the transformation of acid-base property of H3K4 was
likewise ascribed to the variation of the microenvironment. These
results suggest that the neutral state of dimethylated H3K4 was the
dominating form under physiological environment because the
two methyl groups would slightly enhance the hydrophobicity of
the microenvironment and then trigger more effective electrostatic
effect acted on the protonation states. The neutral state of
dimethylated H3K4 agrees with the observation of the neutral
nitrogen at the site of oxidation in other members of monoamine
oxidase family [25,59,60,61,62] and was validated by the
experimental result that LSD1 preferentially bound the substrate
with uncharged dimethylated H3K4 for catalysis . Taken
together, the conservatism of the catalytic microenvironment and
the consistency between theoretical prediction and experimental
results corroborate the deprotonation state of Lys661 and
dimethylated H3K4 under physiological pH conditions. Accord-
ingly, Lys661 and dimethylated H3K4 were kept uncharged
during the following MD simulation and QM/MM calculation.
The 3D model of LSD1-CoREST-substrate complex
Structurally, LSD1 polypeptide chain folds into a highly
asymmetric configuration with three distinct functional domains,
as shown in Figure 3 (middle panel). The N-terminal SWIRM
(Swi3p, Rsc8p and Moira) domain, which is found in several
chromatin-associated proteins, adopts a completely helical histone
fold. It closely packs against the C-terminal AOL (amine oxidase-
like) domain in which the demetylation reaction takes place. The
AOL domain exhibits a significant homologous topology to the
monoamine oxidase family and contains a typical large insertion
that adopts a tower-like structure (Tower domain) with two
antiparallel helices interacting with CoREST.
To investigate the stability of the active site cavity, a 30-ns MD
simulation was performed on the LSD1-CoREST-substrate
complex. The temporal development of the weighted root-mean-
square deviations (RMSD) for the atoms of different domains of
LSD1 and CoREST from their initial positions (t=0) were
monitored. As illustrated in Figure 4, the steady RMSD for the
atoms in the three major functional domains of LSD1, especially
for the catalytic AOL domain, indicated that the catalytic cavity is
relatively stable during the MD simulation and the trajectory of
the MD simulation for the LSD1-CoREST-substrate ternary
complex is reliable for post analysis.
To probe the molecular basis for demeylation catalysis, both
the H-bond and hydrophobic interactions in the active center
were analyzed. In all, the H-bonds amongst LSD1, FAD and
histone substrate were mostly maintained during the 30-ns MD
simulation as indicated by their occupancies (Table S1), which
further indicated the stability of the demethylation environment
during MD simulation. By the distance evolution monitoring of
the region neighboring the flavin ring of FAD, a key lysine was
identified to interact with the N5 atom of FAD via a water
molecule by two H-bonds (Figure 3A), which characterized the
conserved lysine-water-flavin motif. In addition, as shown in
Figure 5, the two H-bonds were mostly conserved during MD
simulation, with an average interaction distance about 3.3 and
3.0 A˚for O(H2O)-N5(FAD) and O(H2O)-NZ(K661), respective-
ly. Therefore, it can be inferred that the stable H-bond
interactions in lysine-water-flavin motif effectively maintained
FAD in a reactive conformation and further facilitated the
biological reaction through polarizing the N5 atom in FAD. This
hypothesis was in agreement with the mutagenesis study that the
disruption of this conserved H-bonds interaction pattern with the
K661A mutation in LSD1 completely abolished its demethylation
activity  and the substitution of the corresponding Lys300
with Met in MPAO resulted in a 1400-fold decrease in the rate of
flavin reduction . Meanwhile, Baron et al  recently
reported that, during the oxidative half-reaction, K661 may
function as a ‘‘entry residue’’ to channel the oxygen molecule to
Table 1. The predicted pKa for K661 and H3K4 in three
distinct states with different internal dielectric constants.
aInternal dielectric constant.
bH3 peptide in bulk solvent.
QM/MM Study of Catalytic Mechanism of LSD1
PLoS ONE | www.plosone.org4September 2011 | Volume 6 | Issue 9 | e25444
the catalytic chamber and contribute to oxygen activation
through stabilizing the superoxide-flavin semiquinone intermedi-
ates, which further highlighted the crucial role of K661 in
demethylation and was compatible with our simulation results.
Furthermore, the hydrophobic interactions between dimethylated
H3K4 and the hydrophobic catalytic chamber (Figure 3B) of
LSD1 were plotted along the simulation time. As shown in
Figure 6, nearly all of the residues in the catalytic chamber were
involved in the hydrophobic interactions with dimethylated
H3K4, viz., Y761, F538, T810, A809, V333, T335, A539 and
Figure 3. The overall structure of LSD1-CoREST-substrate complex and key interactions in the catalytic chamber. Cartoon diagram of
the LSD1-CoREST-substrate complex highlights the SWIRM domain (green), AOL domain (wheat), Tower domain (golden yellow), CoREST (light blue)
and substrate peptide (magenta). (A) H-bond interactions in the conserved lysine-water-flavin motif. The bridging water molecular is shown in red
sphere and the residues and FAD are shown in green and yellow sticks, respectively. H-bonds are indicated with purple dashed lines. (B) Hydrophobic
interactions of dimethylated H3K4 with its surrounding residues in the catalytic chamber. Dimethylated H3K4 is shown in cyan for the sake of clarity.
(C) H-bond interactions of FAD with its surrounding residues. (D) Hydrophobic interactions of FAD with its surrounding residues.
Figure 4. Time dependencies of the weighted root-mean-square deviations for the backbone atoms of the three domains of LSD1
and CoREST from their initial positions during the 30-ns simulation.
QM/MM Study of Catalytic Mechanism of LSD1
PLoS ONE | www.plosone.org5September 2011 | Volume 6 | Issue 9 | e25444
L659, strongly supporting the assumption that the hydrophobic
interaction was a non-trivial driving force for substrate binding
and emphasizing the importance of each residue in the catalytic
chamber for the precise positioning of the methylated lysine with
respect to the flavin ring. This result was fully consistent with the
experimental results that any mutation in catalytic chamber
Figure 5. Distance evolution along simulation time for the two hydrogen bonds in the lysine-water-flavin motif. The red curve and
blue curve were obtained by 5 ps averaged.
Figure 6. The residues involved in hydrophobic interactions with dimethylated H3K4 versus simulation time. Different colors are used
for the sake of clarity.
QM/MM Study of Catalytic Mechanism of LSD1
PLoS ONE | www.plosone.org6 September 2011 | Volume 6 | Issue 9 | e25444
would severely impair the enzyme activity . Remarkably, the
aromatic side chain of Tyr761, which was highly conserved for
consisting of the so-called ‘‘aromatic cage’’ in the monoamine
amine oxidase family [66,67], formed the most stable interactions
with the dimethylamino group of H3K4, implying a more
fundamental role of Tyr761 in positioning the substrate in a
productive binding orientation for catalytic oxidation. In sum,
both the lysine-water-flavin motif and Tyr761 are deemed to be
the crucial factors for maintaining the optimum configurations
between FAD and dimethylated H3K4. The synergy of the two
factors stabilized the catalytic environment and facilitated the
biochemical reaction via both structural and electrostatic effect.
Taken together, the consistence between simulation results and
the experimental data demonstrated that the MD simulation on
LSD1-CoREST-substrate complex is reasonable and our ternary
complex model is reliable for further study.
Sampling from MD simulations
A proper sampling methodology is pivotal to the reliability of
QM/MM calculations. Fraaije MW et al  thoroughly explored
the recurrent features in catalytic apparatus of different flavopro-
teins by carefully inspecting their three-dimensional structures.
They pointed out the essential stereochemistry principles under-
lying the dehydrogenation reactions: (1) the H-bond donor within
the catalytic chamber is located on the flavin side opposite to that
facing the substrate; (2) the angle between N10, N5 and the
hydrogen-bond donor ranges from 116uto 170u; (3) the site of
oxidative attack typically binds in front of the flavin at 3.5 A˚
distance from N5; (4)the angle defined by the site of oxidative
attack with the N5–N10 atoms has a narrow range of 96–117u.
Furthermore, taking into account the extent of fluctuation of
RMSD value during MD simulation, the snapshots in the
equilibrium state since 10 ns were treated with these statistical
criterions. Configurations fulfilling these criterions were extracted
to to select a proper starting point for subsequent QM/MM study.
As shown in Figure S1 in Supporting Information, the binding
mode and conformation of FAD in the selected snapshot closely
resemble those observed in other flavinenzymes [54,68,69]. The
interaction analysis (Figure 3C, 3D) based on the snapshot
indicates the hydrophobic interactions with FAD were highly
conserved and quite compatible with the previous experimental
study , conforming this snapshot was a reasonable initial
structure for the QM/MM study.
Direct hydride transfer mechanism.
underlying feasibility of the reductive half-reaction along the
three debated proposals, the direct hydride transfer mechanism
was firstly investigated with the QM/MM strategy. The system for
aforementioned snapshot. The QM region was composed of the
dimethylamino portion (-(CH2)3-N(CH3)2) of dimethylated H3K4
and the isoalloxazine ring with its adjacent methylene group of
FAD. Furthermore, considering the vital roles of lysine-water-
flavin motif in demethylation, the majority of the side chain (-
(CH2)3-NH2) of Lys661 and the water bridging it with flavin were
also included in the QM region. The remainder of the complex
was included in the MM region. The partitioning scheme for QM
and MM regions is described in the Materials and Methods
section. We designate this structure as a reactant system. ONIOM,
a QM/MM method encoded in Gaussion03, was utilized for all
the QM/MM calculations.
The QM/MM optimized geometry of the reagent system varied
slightly with the initial structure obtained by MD simulation. The
To explore the
constructed based onthe
distance from the oxygen atom of conserved water molecule to the
N5 atom of FAD was 2.93 A˚with that the angle consisted of O-H
and N5 was 175.49u, which indicated a strong H-bond was
involved in the catalytic center. The angle between N10, N5 and
the hydrogen-bond donor was 136.90u. Meanwhile, the site of
oxidative attack bound in front of the flavin at a distance of 3.70 A˚
from N5, and defined the angle with the N5–N10 atoms at
111.42u. The optimized configuration was quite compatible with
the stereochemistry principles for productive binding of flavoen-
zymes , demonstrating that the model was reliable for the
QM/MM calculation. Along the reaction scheme of the direct
hydride transfer mechanism, the energies of the reagent (R),
transition state (TS), and immediate product (P) were determined
by two-dimensional QM/MM potential energy surface by
defining the distances of R(H9-CM) and R(N5-H9) as the reaction
coordinates (Figure 7B,C). In the optimized reagent, R(H9-
CM)=1.11 A˚ and R(N5-H9)=2.88 A˚; while in the optimized
immediate product, theH9-CM
CM)=2.87 A˚) and the N5-H9
H9)=1.05 A˚). The calculated potential energy barrier of Proposal
1 is DE?=32.82 kcal/mol, which is slightly higher than that
of other flavoenzymes, such as MTOX that the calculated
potential energy barrier for hydride transfer mechanism is
about 27.4 kcal/mol.  This higher potential energy barrier is
quite in agreement with the experimental results that the rate
constant for the oxidation of substrate of LSD1 is 2–5 orders of
magnitude slower than values reported for other flavoprotein
The structure of the transition state (TS) in Proposal 1 was
determined by adiabatic mapping at the QM/MM level
H9)=1.2 A˚, and the dimethylamino group tends to be in a plane
concomitant with the contraction of the CM-NT bond. This
structural reorganization clearly illustrated that the N5-H9 bond
was partially formed while the H9-CM bond was partially broken,
and indicated that the reductive half-reaction belonged to the
direct hydride transfer mechanism. The overall reaction is
calculated to be endothermic by DE=5.44 kcal/mol. Further-
more, as the decrease of the distance between N5 and H9, the H9-
CM bond elongated to cleavage spontaneously (Figure S2), the
bond order of CM-NT was transformed from single to double in
the intermediate product, which was in agreement with the direct
hydride transfer mechanism. On the basis of the one-dimensional
potential energy profile (Figure S2), the transition state was located
as R(H9-CM)=1.43 A˚ and R(N5-H9)=1.2 A˚ with the energy
barrier about 32.71 kcal/mol. Accordingly, the results further
proved the reductive half-reaction in the demethylation process
belonged to the direct hydride transfer mechanism, which
coincided with recent findings from the exquisite kinetic isotope
In the redox catalysis, the consumption of the substrates was
often accompanied with the charge transfer and the coinstanta-
neous change of redox states. And during the reductive half-
reaction of LSD1, FAD was reduced to FADH2by a hydride
equivalent that transferred from the dimethylated H3K4 and
resulted in a positive charged imine intermediate. To further
characterize the charge transfer in the reductive half-reaction of
LSD1, the evolution of the electrostatic potential (ESP) derived
charges of all pieces in the QM region were monitored (Figure 8).
As expected, the overall ESP charges of Lys661 and water
molecular remained approximately constant throughout the
reaction, consistent with the fact that they were not directly
involved in the hydride transfer pathway (Figure 2). Whereas the
QM/MM Study of Catalytic Mechanism of LSD1
PLoS ONE | www.plosone.org7September 2011 | Volume 6 | Issue 9 | e25444
apparent proportional variations between the ESP charges of FAD
and substrate were clearly observed. In the reactant system, both
FAD and substrate were uncharged. Then the positive charge on
substrate progressively increased until the formation of the imine
cation intermediate. Meanwhile, the negative charge increasingly
aggregated on FAD which resulted in the reduced cofactor with a
negative net charge. The results definitely indicated that
accompanying with the migration of the hydrogen atom from
CM to N5, a pair of electrons concomitantly transferred from the
substrate to FAD, which was fully compatible with the direct
hydride transfer mechanism and in good agreement with previous
theoretical studies of the catalytic mechanism of D-Amino acid
oxidase (DAAO) .
To investigate the conformational
change of the catalytic region along the polar nucleophilic
mechanism, the NT-C4a bond was constrained and curtailed
from 3.5 to 1.5 A˚, and the full optimization was performed for the
adduct, served as a hinge for the polar nucleophilic mechanism,
was not obtained in our studies. Furthermore, the feasibility of the
(DE?.40 kcal/mol) for the generation of the aminium radical
was predicted to severely impede the following reactions, in good
accordance with the theoretical results of MTOX that an
unreasonable high energy barrier was necessitated for the radical
mechanism . Meanwhile, as a-CH bond cleavage has been
experimentally proven to be the rate-limiting step, there is unlikely
such an unfavorable energy barrier preceded this step. Therefore,
our QM/MM results support the hypothesis that the requisite
intermediates prior to CH bond cleavage for the two mechanisms
could rarely exist due to their unfavorable thermodynamic
Figure 7. The potential energy surface of the reductive half-reaction in the demethylation of LSD1-CoREST-substrate complex. (A)
The QM/MM optimized structures of the reactant (R), transition state (TS), and immediate product (P). For clarity, only the structures in the QM region
have been shown. (B) The potential energy surface (PES) of the reductive half-reaction along the defined reaction coordinates. (C) Contour plot of the
PES corresponding to the central part in (B). The pink triangle line represents the lowest energy pathway on the calculated PES and the position of
the TS is marked by a red triangle.
Figure 8. ESP charge distributions of the four groups in the QM
region along the hydride transfer reaction.
QM/MM Study of Catalytic Mechanism of LSD1
PLoS ONE | www.plosone.org8 September 2011 | Volume 6 | Issue 9 | e25444
properties , and agree well with the experimental evidence
that no intermediates between oxidized and reduced flavin were
detected , providing indirect supports to the direct hydride
LSD1 is the first identified lysine-specific histone demethylase
that can specifically demethylate mono- or dimethylated histone H3
lysine4 (H3-K4) and H3 lysine 9 (H3-K9) via a redox process. It
plays a vital role in cell proliferation, adipogenesis, spermatogenesis,
chromosome segregation and embryonic development. As a
potential drug target for discovering anti-tumor drugs, the medical
importance of this enzyme has also been greatly appreciated.
However, the catalytic mechanism of LSD1 demethylation reaction
remains ambiguous, in particular a heated controversy still lie in the
rate-limiting reductive half-reaction. Therefore, in the present
study, we focused on the reductive half-reaction of the demetylation
reaction in LSD1-CoREST-substrate complex system, and theo-
retically confirmed the catalytic mechanism of this step belongs to
the direct hydride transfer mechanism.
Firstly, by using molecular modeling and theoretical titration
methods, the 3D structural model of LSD1-CoREST-substrate
complex was constructed. Then MD simulations were performed
on the structure model and a representative structure was sampled
from the MD trajectory. The validity of the structural model was
confirmed based on the consistency between simulation results and
available experimental mutagenesis and enzymatic data for LSD1.
The H-bonds and hydrophobic interaction analysis highlighted
the conserved lysine-water-flavin motif and Tyr761 in the catalytic
chamber. The synergy of the two factors stabilized the catalytic
environment and contributed to the optimal orientation between
FAD and dimethylated H3K4. Meanwhile, the pivotal hydrogen
bond interaction pattern in lysine-water-flavin motif was deemed
to facilitate the demethylation reaction by elaborate electrostatic
Secondly, the QM/MM calculations were performed on the
representative complex structure. In addition to the groups directly
participated in the reaction, Lys661 and its bridging water
molecular in the lysine-water-flavin motif were also incorporated
into the QM region in consideration of their non-trivial role in
demetylation reaction. The 2D QM/MM potential energy surface
with the located transition state of R(H9-CM)=1.6 A˚and R(N5-
H9)=1.2 A˚indicated that the reductive half-reaction intrinsically
belongs to the direct hydride transfer mechanism. In addition, this
data was further validated by our charge distribution analysis
along the reaction coordinate. In concert with the absence of
detectable intermediates between oxidized and reduced FAD and
the unfavorable energetics for the other two possible mechanism
proposals, our studies suggested that the rate-limiting reductive
half-reaction of LSD1 employed the direct hydride transfer
mechanism. Accordingly, our research provided a detailed
mechanism elucidation for the reductive half-reaction of demeth-
ylation by LSD1, explored the molecular basis of the demethyl-
ation pathway, and shed light on the discovery of novel
mechanism-based modulators for LSD1.
EST-Substrate complex and their occupancies in the 30-
ns MD simulation.
Hydrogen bonds existing in the LSD1-CoR-
binding site in the superimposed structures obtained
from the sampled snapshot of LSD1 and the crystal
structures of homologous flavinenzymes (1GOS (human
MAO B), 2VVM (Aspergillus niger MAO N) and 1B5Q
(Zea mays PAO)). The carbons in LSD1, 1GOS, 2VVM and
1B5Q are colored by green, cyan, yellow and pink, respectively.
Local conformation of residues around FAD
and the corresponding R(H9-CM) distance profile along
the reaction path obtained by defining the distance of
R(N5-H9) as the reaction coordinate.
The one-dimensional potential energy profile
Conceived and designed the experiments: XK SO BS DL KKL CL HJ.
Performed the experiments: XK SO ZL LC JL KKL CL. Analyzed the
data: XK SO ZL LC JL KKL CL. Wrote the paper: XK SO ZL MZ KKL
1. Berger SL (2007) The complex language of chromatin regulation during
transcription. Nature 447: 407–412.
2. Kouzarides T (2007) Chromatin modifications and their function. Cell 128:
3. Shi Y, Lan F, Matson C, Mulligan P, Whetstine JR, et al. (2004) Histone
demethylation mediated by the nuclear amine oxidase homolog LSD1. Cell 119:
4. Forneris F, Binda C, Vanoni MA, Mattevi A, Battaglioli E (2005) Histone
demethylation catalysed by LSD1 is a flavin-dependent oxidative process. FEBS
Lett 579: 2203–2207.
5. Huang J, Sengupta R, Espejo AB, Lee MG, Dorsey JA, et al. (2007) p53 is
regulated by the lysine demethylase LSD1. Nature 449: 105–108.
6. Tsai WW, Nguyen TT, Shi Y, Barton MC (2008) p53-targeted LSD1 functions
in repression of chromatin structure and transcription in vivo. Mol Cell Biol 28:
7. Wang J, Hevi S, Kurash JK, Lei H, Gay F, et al. (2009) The lysine demethylase
LSD1 (KDM1) is required for maintenance of global DNA methylation. Nat
Genet 41: 125–129.
8. Cho HS, Suzuki T, Dohmae N, Hayami S, Unoki M, et al. (2011)
Demethylation of RB Regulator MYPT1 by Histone Demethylase LSD1
Promotes Cell Cycle Progression in Cancer Cells. Cancer Res 71: 655–660.
9. Wang Y, Zhang H, Chen Y, Sun Y, Yang F, et al. (2009) LSD1 is a subunit of
the NuRD complex and targets the metastasis programs in breast cancer. Cell
10. Lin Y, Wu Y, Li J, Dong C, Ye X, et al. (2010) The SNAG domain of Snail1
functions as a molecular hook for recruiting lysine-specific demethylase 1.
EMBO J 29: 1803–1816.
11. Lee MG, Wynder C, Cooch N, Shiekhattar R (2005) An essential role for
CoREST in nucleosomal histone 3 lysine 4 demethylation. Nature 437: 432–435.
12. Hu X, Li X, Valverde K, Fu X, Noguchi C, et al. (2009) LSD1-mediated
epigenetic modification is required for TAL1 function and hematopoiesis. Proc
Natl Acad Sci U S A 106: 10141–10146.
13. Scoumanne A, Chen X (2007) The lysine-specific demethylase 1 is required for
cell proliferation in both p53-dependent and -independent manners. J Biol
Chem 282: 15471–15475.
14. Musri MM, Carmona MC, Hanzu FA, Kaliman P, Gomis R, et al. (2010)
Histone Demethylase LSD1 Regulates Adipogenesis. Journal of Biological
Chemistry 285: 30034–30041.
15. Godmann M, Auger V, Ferraroni-Aguiar V, Di Sauro A, Sette C, et al. (2007)
Dynamic regulation of histone H3 methylation at lysine 4 in mammalian
spermatogenesis. Biol Reprod 77: 754–764.
16. Lv S, Bu W, Jiao H, Liu B, Zhu L, et al. (2010) LSD1 is required for
chromosome segregation during mitosis. Eur J Cell Biol 89: 557–563.
17. Ding S, Zhou HY, Li WL, Zhu SY, Joo JY, et al. (2010) Conversion of Mouse
Epiblast Stem Cells to an Earlier Pluripotency State by Small Molecules. Journal
of Biological Chemistry 285: 29676–29680.
18. Foster CT, Dovey OM, Lezina L, Luo JL, Gant TW, et al. (2010) Lysine-specific
demethylase 1 regulates the embryonic transcriptome and CoREST stability.
Mol Cell Biol 30: 4851–4863.
19. Schulte JH, Lim S, Schramm A, Friedrichs N, Koster J, et al. (2009) Lysine-
specific demethylase 1 is strongly expressed in poorly differentiated neuroblas-
toma: implications for therapy. Cancer Res 69: 2065–2071.
20. Saramaki A, Diermeier S, Kellner R, Laitinen H, Vaisanen S, et al. (2009)
Cyclical chromatin looping and transcription factor association on the regulatory
QM/MM Study of Catalytic Mechanism of LSD1
PLoS ONE | www.plosone.org9September 2011 | Volume 6 | Issue 9 | e25444
regions of the p21 (CDKN1A) gene in response to 1alpha,25-dihydroxyvitamin
D3. J Biol Chem 284: 8073–8082.
21. Lim S, Janzer A, Becker A, Zimmer A, Schule R, et al. (2010) Lysine-specific
demethylase 1 (LSD1) is highly expressed in ER-negative breast cancers and a
biomarker predicting aggressive biology. Carcinogenesis 31: 512–520.
22. Huang Y, Greene E, Murray Stewart T, Goodwin AC, Baylin SB, et al. (2007)
Inhibition of lysine-specific demethylase 1 by polyamine analogues results in
reexpression of aberrantly silenced genes. Proc Natl Acad Sci U S A 104:
23. Metzger E, Wissmann M, Yin N, Muller JM, Schneider R, et al. (2005) LSD1
demethylates repressive histone marks to promote androgen-receptor-dependent
transcription. Nature 437: 436–439.
24. Kahl P, Gullotti L, Heukamp LC, Wolf S, Friedrichs N, et al. (2006) Androgen
receptor coactivators lysine-specific histone demethylase 1 and four and a half
LIM domain protein 2 predict risk of prostate cancer recurrence. Cancer Res 66:
25. Kurtz KA, Rishavy MA, Cleland WW, Fitzpatrick PF (2000) Nitrogen isotope
effects as probes of the mechanism of D-amino acid oxidase. Journal of the
American Chemical Society 122: 12896–12897.
26. Edmondson DE, Binda C, Mattevi A (2007) Structural insights into the
mechanism of amine oxidation by monoamine oxidases A and B. Archives of
Biochemistry and Biophysics 464: 269–276.
27. Silverman RB (1995) Radical Ideas About Monoamine-Oxidase. Accounts of
Chemical Research 28: 335–342.
28. Yang M, Gocke CB, Luo X, Borek D, Tomchick DR, et al. (2006) Structural
basis for CoREST-dependent demethylation of nucleosomes by the human
LSD1 histone demethylase. Mol Cell 23: 377–387.
29. Forneris F, Binda C, Adamo A, Battaglioli E, Mattevi A (2007) Structural basis
of LSD1-CoREST selectivity in histone H3 recognition. J Biol Chem 282:
30. Gordon JC, Myers JB, Folta T, Shoja V, Heath LS, et al. (2005) H++: a server
for estimating pKas and adding missing hydrogens to macromolecules. Nucleic
Acids Res 33: W368–371.
31. Bayly CI, Cieplak P, Cornell WD, Kollman PA (1993) A Well-Behaved
Electrostatic Potential Based Method Using Charge Restraints for Deriving
Atomic Charges - the Resp Model. Journal of Physical Chemistry 97:
32. Case DA, Cheatham TE, 3rd, Darden T, Gohlke H, Luo R, et al. (2005) The
Amber biomolecular simulation programs. J Comput Chem 26: 1668–1688.
33. Cornell WD, Cieplak P, Bayly CI, Gould IR, Merz KM, et al. (1996) A second
generation force field for the simulation of proteins, nucleic acids, and organic
molecules (vol 117, pg 5179, 1995). Journal of the American Chemical Society
34. Wang JM, Cieplak P, Kollman PA (2000) How well does a restrained
electrostatic potential (RESP) model perform in calculating conformational
energies of organic and biological molecules? Journal of Computational
Chemistry 21: 1049–1074.
35. Hornak V, Abel R, Okur A, Strockbine B, Roitberg A, et al. (2006) Comparison
of multiple Amber force fields and development of improved protein backbone
parameters. Proteins 65: 712–725.
36. Jorgensen WL, Chandrasekhar J, Madura JD, Impey RW, Klein ML (1983)
Comparison of Simple Potential Functions for Simulating Liquid Water. Journal
of Chemical Physics 79: 926–935.
37. Ryckaert JP, Ciccotti G, Berendsen HJC (1977) Numerical-Integration of
Cartesian Equations of Motion of a System with Constraints - Molecular-
Dynamics of N-Alkanes. Journal of Computational Physics 23: 327–341.
38. Darden T, York D, Pedersen L (1993) Particle Mesh Ewald - an N.Log(N)
Method for Ewald Sums in Large Systems. Journal of Chemical Physics 98:
39. Berendsen HJC, Postma JPM, van Gunsteren WF, Dinola A, Haak JR (1984)
Molecular-Dynamics with Coupling to an External Bath. Journal of Chemical
Physics 81: 3684–3690.
40. Frisch MJ Gaussian 03, revision B.05 Gaussian, Inc.
41. Maseras F, Morokuma K (1995) Imomm - a New Integrated Ab-Initio Plus
Molecular Mechanics Geometry Optimization Scheme of Equilibrium Struc-
tures and Transition-States. Journal of Computational Chemistry 16:
42. Svensson M, Humbel S, Morokuma K (1996) Energetics using the single point
IMOMO (integrated molecular orbital plus molecular orbital) calculations:
Choices of computational levels and model system. Journal of Chemical Physics
43. Dapprich S, Komaromi I, Byun KS, Morokuma K, Frisch MJ (1999) A new
ONIOM implementation in Gaussian98. Part I. The calculation of energies,
gradients, vibrational frequencies and electric field derivatives. Journal of
Molecular Structure-Theochem 461: 1–21.
44. Vreven T, Morokuma K (2000) On the application of the IMOMO (integrated
molecular orbital plus molecular orbital) method. Journal of Computational
Chemistry 21: 1419–1432.
45. Vreven T, Mennucci B, da Silva CO, Morokuma K, Tomasi J (2001) The
ONIOM-PCM method: Combining the hybrid molecular orbital method and
the polarizable continuum model for solvation. Application to the geometry and
properties of a merocyanine in solution. Journal of Chemical Physics 115: 62–72.
46. Schlegel HB, Vreven T, Morokuma K, Farkas O, Frisch MJ (2003) Geometry
optimization with QM/MM, ONIOM, and other combined methods. I.
Microiterations and constraints. Journal of Computational Chemistry 24:
47. Liang Z, Shi T, Ouyang S, Li H, Yu K, et al. (2010) Investigation of the catalytic
mechanism of Sir2 enzyme with QM/MM approach: SN1 vs SN2? Journal of
Physical Chemistry B 114: 11927–11933.
48. Field MJ, Bash PA, Karplus M (1990) A Combined Quantum-Mechanical and
Molecular Mechanical Potential for Molecular-Dynamics Simulations. Journal
of Computational Chemistry 11: 700–733.
49. Singh UC, Kollman PA (1986) A Combined Abinitio Quantum-Mechanical and
Molecular Mechanical Method for Carrying out Simulations on Complex
Molecular-Systems - Applications to the Ch3cl+Cl2 Exchange-Reaction and
Gas-Phase Protonation of Polyethers. Journal of Computational Chemistry 7:
50. Raghavachari K (2000) Perspective on ‘‘Density functional thermochemistry.
III. The role of exact exchange’’ - Becke AD (1993) J Chem Phys 98:5648–52.
Theoretical Chemistry Accounts 103: 361–363.
51. Lee CT, Yang WT, Parr RG (1988) Development of the Colle-Salvetti
Correlation-Energy Formula into a Functional of the Electron-Density. Physical
Review B 37: 785–789.
52. Vosko SH, Wilk L, Nusair M (1980) Accurate Spin-Dependent Electron Liquid
Correlation Energies for Local Spin-Density Calculations - a Critical Analysis.
Canadian Journal of Physics 58: 1200–1211.
53. Stephens PJ, Devlin FJ, Chabalowski CF, Frisch MJ (1994) Ab-Initio Calculation
of Vibrational Absorption and Circular-Dichroism Spectra Using Density-
Functional Force-Fields. Journal of Physical Chemistry 98: 11623–11627.
54. Binda C, Coda A, Angelini R, Federico R, Ascenzi P, et al. (1999) A 30
angstrom long U-shaped catalytic tunnel in the crystal structure of polyamine
oxidase. Structure with Folding & Design 7: 265–276.
55. Kim JJP, Wang M, Paschke R (1993) Crystal-Structures of Medium-Chain Acyl-
Coa Dehydrogenase from Pig-Liver Mitochondria with and without Substrate.
Proceedings of the National Academy of Sciences of the United States of
America 90: 7523–7527.
56. Fraaije MW, Mattevi A (2000) Flavoenzymes: diverse catalysts with recurrent
features. Trends in Biochemical Sciences 25: 126–132.
57. Polticelli F, Basran J, Faso C, Cona A, Minervini G, et al. (2005) Lys300 plays a
major role in the catalytic mechanism of maize polyamine oxidase. Biochemistry
58. Pozzi MH, Fitzpatrick PF (2010) A lysine conserved in the monoamine oxidase
family is involved in oxidation of the reduced flavin in mouse polyamine oxidase.
Archives of Biochemistry and Biophysics 498: 83–88.
59. Pozzi MH, Gawandi V, Fitzpatrick PF (2009) pH Dependence of a Mammalian
Polyamine Oxidase: Insights into Substrate Specificity and the Role of Lysine
315. Biochemistry 48: 1508–1516.
60. Ralph EC, Anderson MA, Cleland WW, Fitzpatrick PF (2006) Mechanistic
studies of the flavoenzyme tryptophan 2-monooxygenase: Deuterium and N-15
kinetic isotope effects on alanine oxidation by an L-amino acid oxidase.
Biochemistry 45: 15844–15852.
61. Ralph EC, Fitzpatrick PF (2005) pH and kinetic isotope effects on sarcosine
oxidation by N-methyltryptophan oxidase. Biochemistry 44: 3074–3081.
62. Dunn RV, Marshall KR, Munro AW, Scrutton NS (2008) The pH dependence
of kinetic isotope effects in monoamine oxidase A indicates stabilization of the
neutral amine in the enzyme-substrate complex. Febs Journal 275: 3850–3858.
63. Gaweska H, Henderson Pozzi M, Schmidt DM, McCafferty DG, Fitzpatrick PF
(2009) Use of pH and kinetic isotope effects to establish chemistry as rate-limiting
in oxidation of a peptide substrate by LSD1. Biochemistry 48: 5440–5445.
64. Stavropoulos P, Blobel G, Hoelz A (2006) Crystal structure and mechanism of
human lysine-specific demethylase-1. Nature Structural & Molecular
Biology 13: 626–632.
65. Baron R, Binda C, Tortorici M, McCammon JA, Mattevi A (2011) Molecular
Mimicry and Ligand Recognition in Binding and Catalysis by the Histone
Demethylase LSD1-CoREST Complex. Structure 19: 212–220.
66. Binda C, Mattevi A, Edmondson DE (2002) Structure-function relationships in
flavoenzyme-dependent amine oxidations: a comparison of polyamine oxidase
and monoamine oxidase. J Biol Chem 277: 23973–23976.
67. Forneris F, Battaglioli E, Mattevi A, Binda C (2009) New roles of flavoproteins in
molecular cell biology: histone demethylase LSD1 and chromatin. FEBS J 276:
68. Binda C, Newton-Vinson P, Hubalek F, Edmondson DE, Mattevi A (2002)
Structure of human monoamine oxidase B, a drug target for the treatment of
neurological disorders. Nat Struct Biol 9: 22–26.
69. Wierenga RK, Drenth J, Schulz GE (1983) Comparison of the three-
dimensional protein and nucleotide structure of the FAD-binding domain of
p-hydroxybenzoate hydroxylase with the FAD- as well as NADPH-binding
domains of glutathione reductase. J Mol Biol 167: 725–739.
70. Chen Y, Yang Y, Wang F, Wan K, Yamane K, et al. (2006) Crystal structure of
human histone lysine-specific demethylase 1 (LSD1). Proc Natl Acad Sci U S A
71. Ralph EC, Hirschi JS, Anderson MA, Cleland WW, Singleton DA, et al. (2007)
Insights into the mechanism of flavoprotein-catalyzed amine oxidation from
nitrogen isotope effects on the reaction of N-methyltryptophan oxidase.
Biochemistry 46: 7655–7664.
72. Ralph EC, Anderson MA, Cleland WW, Fitzpatrick PF (2006) Mechanistic
studies of the flavoenzyme tryptophan 2-monooxygenase: deuterium and 15N
QM/MM Study of Catalytic Mechanism of LSD1
PLoS ONE | www.plosone.org 10September 2011 | Volume 6 | Issue 9 | e25444
kinetic isotope effects on alanine oxidation by an L-amino acid oxidase.
Biochemistry 45: 15844–15852.
73. Sobrado P, Fitzpatrick PF (2003) Solvent and primary deuterium isotope effects
show that lactate CH and OH bond cleavages are concerted in Y254F
flavocytochrome b(2), consistent with a hydride transfer mechanism. Biochem-
istry 42: 15208–15214.
74. Ghanem M, Gadda G (2005) On the catalytic role of the conserved active site
residue His466 of choline oxidase. Biochemistry 44: 893–904.
75. Brinkley DW, Roth JP (2005) Determination of a large reorganization energy
barrier for hydride abstraction by glucose oxidase. J Am Chem Soc 127:
76. Menon V, Hsieh CT, Fitzpatrick PF (1995) Substituted Alcohols as Mechanistic
Probes of Alcohol Oxidase. Bioorganic Chemistry 23: 42–53.
77. Pozzi MH, Gawandi V, Fitzpatrick PF (2009) Mechanistic Studies of para-
Substituted N,N9-Dibenzyl-1,4-diaminobutanes as Substrates for a Mammalian
Polyamine Oxidase. Biochemistry 48: 12305–12313.
78. Yuan HL, Gadda G (2011) Importance of a Serine Proximal to the C(4a) and
N(5) Flavin Atoms for Hydride Transfer in Choline Oxidase. Biochemistry 50:
79. Tilocca A, Gamba A, Vanoni MA, Fois E (2002) First-principles molecular
dynamics investigation of the D-amino acid oxidative half-reaction catalyzed by
the flavoenzyme D-amino acid oxidase. Biochemistry 41: 14111–14121.
80. Fitzpatrick PF (2010) Oxidation of amines by flavoproteins. Archives of
Biochemistry and Biophysics 493: 13–25.
QM/MM Study of Catalytic Mechanism of LSD1
PLoS ONE | www.plosone.org 11September 2011 | Volume 6 | Issue 9 | e25444