26 research outputs found
Design, Synthesis, and Evaluation of Polyamine Deacetylase Inhibitors, and High-Resolution Crystal Structures of Their Complexes with Acetylpolyamine Amidohydrolase
Polyamines are essential aliphatic
polycations that bind to nucleic
acids and accordingly are involved in a variety of cellular processes.
Polyamine function can be regulated by acetylation and deacetylation,
just as histone function can be regulated by lysine acetylation and
deacetylation. Acetylpolyamine amidohydrolase (APAH) from <i>Mycoplana ramosa</i> is a zinc-dependent polyamine deacetylase
that shares approximately 20% amino acid sequence identity with human
histone deacetylases. We now report the X-ray crystal structures of
APAHâinhibitor complexes in a new and superior crystal form
that diffracts to very high resolution (1.1â1.4 Ă
). Inhibitors
include previously synthesized analogues of <i>N</i><sup>8</sup>-acetylspermidine bearing trifluoromethylketone, thiol, and
hydroxamate zinc-binding groups [Decroos, C., Bowman, C. M., and Christianson,
D. W. (2013) <i>Bioorg. Med. Chem. 21</i>, 4530], and newly
synthesized hydroxamate analogues of shorter, monoacetylated diamines,
the most potent of which is the hydroxamate analogue of <i>N</i>-acetylcadaverine (IC<sub>50</sub> = 68 nM). The high-resolution
crystal structures of APAHâinhibitor complexes provide key
inferences about the inhibition and catalytic mechanism of zinc-dependent
deacetylases. For example, the trifluoromethylketone analogue of <i>N</i><sup>8</sup>-acetylspermidine binds as a tetrahedral gem-diol
that mimics the tetrahedral intermediate and its flanking transition
states in catalysis. Surprisingly, this compound is also a potent
inhibitor of human histone deacetylase 8 with an IC<sub>50</sub> of
260 nM. Crystal structures of APAHâinhibitor complexes are
determined at the highest resolution of any currently existing zinc
deacetylase structure and thus represent the most accurate reference
points for understanding structureâmechanism and structureâinhibition
relationships in this critically important enzyme family
Structure-Based Engineering of a Sesquiterpene Cyclase to Generate an Alcohol Product: Conversion of <i>epi</i>-Isozizaene Synthase into 뱉Bisabolol Synthase
The sesquiterpene cyclase epi-isozizaene
synthase
(EIZS) from Streptomyces coelicolor catalyzes the metal-dependent conversion of farnesyl diphosphate
(FPP) into the complex tricyclic product epi-isozizaene.
This remarkable transformation is governed by an active site contour
that serves as a template for catalysis, directing the conformations
of multiple carbocation intermediates leading to the final product.
Mutagenesis of residues defining the active site contour remolds its
three-dimensional shape and reprograms the cyclization cascade to
generate alternative cyclization products. In some cases, mutagenesis
enables alternative chemistry to quench carbocation intermediates,
e.g., through hydroxylation. Here, we combine structural and biochemical
data from previously characterized EIZS mutants to design and prepare
F95SâF198S EIZS, which converts EIZS into an α-bisabolol
synthase with moderate fidelity (65% at 18 °C, 74% at 4 °C).
We report the complete biochemical characterization of this double
mutant as well as the 1.47 Ă
resolution X-ray crystal structure
of its complex with three Mg2+ ions, inorganic pyrophosphate,
and the benzyltriethylammonium cation, which partially mimics a carbocation
intermediate. Most notably, the two mutations together create an active
site contour that stabilizes the bisabolyl carbocation intermediate
and positions a water molecule for the hydroxylation reaction. Structural
comparison with a naturally occurring α-bisabolol synthase reveals
common active site features that direct α-bisabolol generation.
In showing that EIZS can be redesigned to generate a sesquiterpene
alcohol product instead of a sesquiterpene hydrocarbon product, we
have expanded the potential of EIZS as a platform for the development
of designer cyclases that could be utilized in synthetic biology applications
Binding of the Microbial Cyclic Tetrapeptide Trapoxin A to the Class I Histone Deacetylase HDAC8
Trapoxin
A is a microbial cyclic tetrapeptide that is an essentially
irreversible inhibitor of class I histone deacetylases (HDACs). The
inhibitory warhead is the α,ÎČ-epoxyketone side-chain of
(2<i>S</i>,9<i>S</i>)-2-amino-8-oxo-9,10-epoxydecanoic
acid (l-Aoe), which mimics the side-chain of the HDAC substrate
acetyl-l-lysine. We now report the crystal structure of the
HDAC8âtrapoxin A complex at 1.24 Ă
resolution, revealing
that the ketone moiety of l-Aoe undergoes nucleophilic attack
to form a zinc-bound tetrahedral gem-diolate that mimics the tetrahedral
intermediate and its flanking transition states in catalysis. Mass
spectrometry, activity measurements, and isothermal titration calorimetry
confirm that trapoxin A binds tightly (<i>K</i><sub>d</sub> = 3 ± 1 nM) and does not covalently modify the enzyme, so the
epoxide moiety of l-Aoe remains intact. Comparison of the
HDAC8âtrapoxin A complex with the HDAC6-HC toxin complex provides
new insight regarding the inhibitory potency of l-Aoe-containing
natural products against class I and class II HDACs
Entropy as a Driver of Selectivity for Inhibitor Binding to Histone Deacetylase 6
Among the metal-dependent histone
deacetylases, the class IIb isozyme
HDAC6 is remarkable because of its role in the regulation of microtubule
dynamics in the cytosol. Selective inhibition of HDAC6 results in
microtubule hyperacetylation, leading to cell cycle arrest and apoptosis,
which is a validated strategy for cancer chemotherapy and the treatment
of other disorders. HDAC6 inhibitors generally consist of a Zn<sup>2+</sup>-binding group such as a hydroxamate, a linker, and a capping
group; the capping group is a critical determinant of isozyme selectivity.
Surprisingly, however, even âcaplessâ inhibitors exhibit
appreciable HDAC6 selectivity. To probe the chemical basis for this
selectivity, we now report high-resolution crystal structures of HDAC6
complexed with capless cycloalkyl hydroxamate inhibitors <b>1â4</b>. Each inhibitor hydroxamate group coordinates to the catalytic Zn<sup>2+</sup> ion with canonical bidentate geometry. Additionally, the
olefin moieties of compounds <b>2</b> and <b>4</b> bind
in an aromatic crevice between the side chains of F583 and F643. Reasoning
that similar binding could be achieved in the representative class
I isozyme HDAC8, we employed isothermal titration calorimetry to study
the thermodynamics of inhibitor binding. These measurements indicate
that the entropy of inhibitor binding is generally positive for binding
to HDAC6 and negative for binding to HDAC8, resulting in â€313-fold
selectivity for binding to HDAC6 relative to HDAC8. Thus, favorable
binding entropy contributes to HDAC6 selectivity. Notably, cyclohexenyl
hydroxamate <b>2</b> represents a promising lead for derivatization
with capping groups that may further enhance its impressive 313-fold
thermodynamic selectivity for HDAC6 inhibition
Structure of 2-Methylisoborneol Synthase from <i>Streptomyces coelicolor</i> and Implications for the Cyclization of a Noncanonical <i>C</i>-Methylated Monoterpenoid Substrate
The crystal structure of 2-methylisoborneol synthase
(MIBS) from <i>Streptomyces coelicolor</i> A3(2) has been
determined in complex with substrate analogues geranyl-<i>S</i>-thiolodiphosphate and 2-fluorogeranyl diphosphate at 1.80 and 1.95
Ă
resolution, respectively. This terpenoid cyclase catalyzes
the cyclization of the naturally occurring, noncanonical <i>C</i>-methylated isoprenoid substrate, 2-methylgeranyl diphosphate, to
form the bicyclic product 2-methylisoborneol, a volatile C<sub>11</sub> homoterpene alcohol with an earthy, musty odor. While MIBS adopts
the tertiary structure of a class I terpenoid cyclase, its dimeric
quaternary structure differs from that previously observed in dimeric
terpenoid cyclases from plants and fungi. The quaternary structure
of MIBS is nonetheless similar in some respects to that of dimeric
farnesyl diphosphate synthase, which is not a cyclase. The structures
of MIBS complexed with substrate analogues provide insights regarding
differences in the catalytic mechanism of MIBS and the mechanisms
of (+)-bornyl diphosphate synthase and <i>endo</i>-fenchol
synthase, plant cyclases that convert geranyl diphosphate into products
with closely related bicyclic bornyl skeletons, but distinct structures
and stereochemistries
Unexpected Reactivity of 2âFluorolinalyl Diphosphate in the Active Site of Crystalline 2âMethylisoborneol Synthase
The
crystal structure of 2-methylisoborneol synthase (MIBS) from <i>Streptomyces coelicolor</i> A3(2) has been determined in its
unliganded state and in complex with two Mg<sup>2+</sup> ions and
2-fluoroneryl diphosphate at 1.85 and 2.00 Ă
resolution, respectively.
Under normal circumstances, MIBS catalyzes the cyclization of the
naturally occurring, noncanonical 11-carbon isoprenoid substrate,
2-methylgeranyl diphosphate, which first undergoes an ionizationâisomerizationâionization
sequence through the tertiary diphosphate intermediate 2-methyllinalyl
diphosphate to enable subsequent cyclization chemistry. MIBS does
not exhibit catalytic activity with 2-fluorogeranyl diphosphate, and
we recently reported the crystal structure of MIBS complexed with
this unreactive substrate analogue [KoÌksal, M., Chou, W. K. W., Cane, D. E., Christianson, D.
W. (2012) Biochemistry 51, 3011â3020]. However, cocrystallization of MIBS with the fluorinated analogue
of the tertiary allylic diphosphate intermediate, 2-fluorolinalyl
diphosphate, reveals unexpected reactivity for the intermediate analogue
and yields the crystal structure of the complex with the primary allylic
diphosphate, 2-fluoroneryl diphosphate. Comparison with the structure
of the unliganded enzyme reveals that the crystalline enzyme active
site remains partially open, presumably due to the binding of only
two Mg<sup>2+</sup> ions. Assays in solution indicate that MIBS catalyzes
the generation of (1<i>R</i>)-(+)-camphor from the substrate
2-fluorolinalyl diphosphate, suggesting that both 2-fluorolinalyl
diphosphate and 2-methyllinalyl diphosphate follow the identical cyclization
mechanism leading to 2-substituted isoborneol products; however, the
initially generated 2-fluoroisoborneol cyclization product is unstable
and undergoes elimination of hydrogen fluoride to yield (1<i>R</i>)-(+)-camphor
Structure of Geranyl Diphosphate <i>C</i>-Methyltransferase from <i>Streptomyces coelicolor</i> and Implications for the Mechanism of Isoprenoid Modification
Geranyl diphosphate <i>C</i>-methyltransferase
(GPPMT)
from <i>Streptomyces coelicolor</i> A3(2) is the first methyltransferase
discovered that modifies an acyclic isoprenoid diphosphate, geranyl
diphosphate (GPP), to yield a noncanonical acyclic allylic diphosphate
product, 2-methylgeranyl diphosphate, which serves as the substrate
for a subsequent cyclization reaction catalyzed by a terpenoid cyclase,
methylisoborneol synthase. Here, we report the crystal structures
of GPPMT in complex with GPP or the substrate analogue geranyl <i>S</i>-thiolodiphosphate (GSPP) along with <i>S</i>-adenosyl-l-homocysteine in the cofactor binding site, resulting
from <i>in situ</i> demethylation of <i>S</i>-adenosyl-l-methionine, at 2.05 or 1.82 Ă
resolution, respectively.
These structures suggest that both GPP and GSPP can undergo catalytic
methylation in crystalline GPPMT, followed by dissociation of the
isoprenoid product. <i>S</i>-Adenosyl-l-homocysteine
remains bound in the active site, however, and does not exchange with
a fresh molecule of cofactor <i>S</i>-adenosyl-l-methionine. These structures provide important clues about the molecular
mechanism of the reaction, especially with regard to the face of the
2,3 double bond of GPP that is methylated as well as the stabilization
of the resulting carbocation intermediate through cationâÏ
interactions
Crystal Structure of an Arginase-like Protein from <i>Trypanosoma brucei</i> That Evolved without a Binuclear Manganese Cluster
The X-ray crystal structure of an
arginase-like protein from the
parasitic protozoan <i>Trypanosoma brucei</i>, designated
TbARG, is reported at 1.80 and 2.38 Ă
resolution in its reduced
and oxidized forms, respectively. The oxidized form of TbARG is a
disulfide-linked hexamer that retains the overall architecture of
a dimer of trimers in the reduced form. Intriguingly, TbARG does not
contain metal ions in its putative active site, and amino acid sequence
comparisons indicate that all but one of the residues required for
coordination to the catalytically obligatory binuclear manganese cluster
in other arginases are substituted here with residues incapable of
metal ion coordination. Therefore, the structure of TbARG is the first
of a member of the arginase/deacetylase superfamily that is not a
metalloprotein. Although we show that metal binding activity is easily
reconstituted in TbARG by site-directed mutagenesis and confirmed
in X-ray crystal structures, it is curious that this protein and its
parasitic orthologues evolved away from metal binding function. Knockout
of the TbARG gene from the genome demonstrated that its function is
not essential to cultured bloodstream-form <i>T. brucei</i>, and metabolomics analysis confirmed that the enzyme has no role
in the conversion of l-arginine to l-ornithine in
these cells. While the molecular function of TbARG remains enigmatic,
the fact that the <i>T. brucei</i> genome encodes only this
protein and not a functional arginase indicates that the parasite
must import l-ornithine from its host to provide a source
of substrate for ornithine decarboxylase in the polyamine biosynthetic
pathway, an active target for the development of antiparasitic drugs
Structure and Function of Fusicoccadiene Synthase, a Hexameric Bifunctional Diterpene Synthase
Fusicoccin
A is a diterpene glucoside phytotoxin generated by the
fungal pathogen <i>Phomopsis amygdali</i> that causes the
plant disease constriction canker, first discovered in New Jersey
peach orchards in the 1930s. Fusicoccin A is also an emerging new
lead in cancer chemotherapy. The hydrocarbon precursor of fusicoccin
A is the tricyclic diterpene fusicoccadiene, which is generated by
a bifunctional terpenoid synthase. Here, we report X-ray crystal structures
of the individual catalytic domains of fusicoccadiene synthase: the
C-terminal domain is a chain elongation enzyme that generates geranylgeranyl
diphosphate, and the N-terminal domain catalyzes the cyclization of
geranylgeranyl diphosphate to form fusicoccadiene. Crystal structures
of each domain complexed with bisphosphonate substrate analogues suggest
that three metal ions and three positively charged amino acid side
chains trigger substrate ionization in each active site. While <i>in vitro</i> incubations reveal that the cyclase domain can
utilize farnesyl diphosphate and geranyl diphosphate as surrogate
substrates, these shorter isoprenoid diphosphates are mainly converted
into acyclic alcohol or hydrocarbon products. Gel filtration chromatography
and analytical ultracentrifugation experiments indicate that full-length
fusicoccadiene synthase adopts hexameric quaternary structure, and
small-angle X-ray scattering data yield a well-defined molecular envelope
illustrating a plausible model for hexamer assembly
Biochemical and Structural Characterization of HDAC8 Mutants Associated with Cornelia de Lange Syndrome Spectrum Disorders
Cornelia de Lange Syndrome (CdLS)
spectrum disorders are characterized
by multiple organ system congenital anomalies that result from mutations
in genes encoding core cohesin proteins SMC1A, SMC3, and RAD21, or
proteins that regulate cohesin function such as NIPBL and HDAC8. HDAC8
is the Zn<sup>2+</sup>-dependent SMC3 deacetylase required for cohesin
recycling during the cell cycle, and 17 different HDAC8 mutants have
been identified to date in children diagnosed with CdLS. As part of
our continuing studies focusing on aberrant HDAC8 function in CdLS,
we now report the preparation and biophysical evaluation of five human
HDAC8 mutants: P91L, G117E, H180R, D233G, and G304R. Additionally,
the double mutants D233GâY306F and P91LâY306F were prepared
to enable cocrystallization of intact enzymeâsubstrate complexes.
X-ray crystal structures of G117E, P91LâY306F, and D233GâY306F
HDAC8 mutants reveal that each CdLS mutation causes structural changes
that compromise catalysis and/or thermostability. For example, the
D233G mutation disrupts the D233âK202âS276 hydrogen
bond network, which stabilizes key tertiary structure interactions,
thereby significantly compromising thermostability. Molecular dynamics
simulations of H180R and G304R HDAC8 mutants suggest that the bulky
arginine side chain of each mutant protrudes into the substrate binding
site and also causes active site residue Y306 to fluctuate away from
the position required for substrate activation and catalysis. Significantly,
the catalytic activities of most mutants can be partially or fully
rescued by the activator <i>N</i>-(phenylcarbamothioyl)-benzamide,
suggesting that HDAC8 activators may serve as possible leads in the
therapeutic management of CdLS