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 APAH–inhibitor complexes in a new and superior crystal form that diffracts to very high resolution (1.1–1.4 Å). Inhibitors include previously synthesized analogues of <i>N</i>⁸-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 (IC₅₀ = 68 nM). The high-resolution crystal structures of APAH–inhibitor complexes provide key inferences about the inhibition and catalytic mechanism of zinc-dependent deacetylases. For example, the trifluoromethylketone analogue of <i>N</i>⁸-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 IC₅₀ of 260 nM. Crystal structures of APAH–inhibitor complexes are determined at the highest resolution of any currently existing zinc deacetylase structure and thus represent the most accurate reference points for understanding structure–mechanism and structure–inhibition relationships in this critically important enzyme family

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,β-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 HDAC8–trapoxin A complex at 1.24 Å 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>_d = 3 ± 1 nM) and does not covalently modify the enzyme, so the epoxide moiety of l-Aoe remains intact. Comparison of the HDAC8–trapoxin 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 Zn²⁺-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 “capless” 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>1–4</b>. Each inhibitor hydroxamate group coordinates to the catalytic Zn²⁺ 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 ≤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 Å 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 C₁₁ 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 2‑Fluorolinalyl Diphosphate in the Active Site of Crystalline 2‑Methylisoborneol 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 Mg²⁺ ions and 2-fluoroneryl diphosphate at 1.85 and 2.00 Å 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 ionization–isomerization–ionization 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 [Köksal, M., Chou, W. K. W., Cane, D. E., Christianson, D. W. (2012) Biochemistry 51, 3011–3020]. 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 Mg²⁺ 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 Å 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 cation−π 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 Å 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 Zn²⁺-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 D233G–Y306F and P91L–Y306F were prepared to enable cocrystallization of intact enzyme–substrate complexes. X-ray crystal structures of G117E, P91L–Y306F, and D233G–Y306F HDAC8 mutants reveal that each CdLS mutation causes structural changes that compromise catalysis and/or thermostability. For example, the D233G mutation disrupts the D233–K202–S276 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

Compromised Structure and Function of HDAC8 Mutants Identified in Cornelia de Lange Syndrome Spectrum Disorders

Cornelia de Lange Syndrome (CdLS) is a multiple congenital anomaly disorder resulting from mutations in genes that encode the core components of the cohesin complex, SMC1A, SMC3, and RAD21, or two of its regulatory proteins, NIPBL and HDAC8. HDAC8 is the human SMC3 lysine deacetylase required for cohesin recycling in the cell cycle. To date, 16 different missense mutations in HDAC8 have recently been identified in children diagnosed with CdLS. To understand the molecular effects of these mutations in causing CdLS and overlapping phenotypes, we have fully characterized the structure and function of five HDAC8 mutants: C153F, A188T, I243N, T311M, and H334R. X-ray crystal structures reveal that each mutation causes local structural changes that compromise catalysis and/or thermostability. For example, the C153F mutation triggers conformational changes that block acetate product release channels, resulting in only 2% residual catalytic activity. In contrast, the H334R mutation causes structural changes in a polypeptide loop distant from the active site and results in 91% residual activity, but the thermostability of this mutant is significantly compromised. Strikingly, the catalytic activity of these mutants can be partially or fully rescued <i>in vitro</i> by the HDAC8 activator <i>N</i>-(phenylcarbamothioyl)­benzamide. These results suggest that HDAC8 activators might be useful leads in the search for new therapeutic strategies in managing CdLS