Integrated Experimental and Theoretical Investigation of Copper Active Site Properties of a Lytic Polysaccharide Monooxygenase from Serratia marcescens.

In this paper, we employed a multidisciplinary approach, combining experimental techniques and density functional theory (DFT) calculations to elucidate key features of the copper coordination environment of the bacterial lytic polysaccharide monooxygenase (LPMO) from Serratia marcescens (SmAA10). The structure of the holo-enzyme was successfully obtained by X-ray crystallography. We then determined the copper(II) binding affinity using competing ligands and observed that the affinity of the histidine brace ligands for copper is significantly higher than previously described. UV-vis, advanced electron paramagnetic resonance (EPR), and X-ray absorption spectroscopy (XAS) techniques, including high-energy resolution fluorescence detected (HERFD) XAS, were further used to gain insight into the copper environment in both the Cu(II) and Cu(I) redox states. The experimental data were successfully rationalized by DFT models, offering valuable information on the electronic structure and coordination geometry of the copper center. Finally, the Cu(II)/Cu(I) redox potential was determined using two different methods at ca. 350 mV vs NHE and rationalized by DFT calculations. This integrated approach not only advances our knowledge of the active site properties of SmAA10 but also establishes a robust framework for future studies of similar enzymatic systems.

Decoding the Ambiguous Electron Paramagnetic Resonance Signals in the Lytic Polysaccharide Monooxygenase from Photorhabdus luminescens.

Understanding the structure and function of lytic polysaccharide monooxygenases (LPMOs), copper enzymes that degrade recalcitrant polysaccharides, requires the reliable atomistic interpretation of electron paramagnetic resonance (EPR) data on the Cu(II) active site. Among various LPMO families, the chitin-active PlAA10 shows an intriguing phenomenology with distinct EPR signals, a major rhombic and a minor axial signal. Here, we combine experimental and computational investigations to uncover the structural identity of these signals. X-band EPR spectra recorded at different pH values demonstrate pH-dependent population inversion: the major rhombic signal at pH 6.5 becomes minor at pH 8.5, where the axial signal dominates. This suggests that a protonation change is involved in the interconversion. Precise structural interpretations are pursued with quantum chemical calculations. Given that accurate calculations of Cu g-tensors remain challenging for quantum chemistry, we first address this problem via a thorough calibration study. This enables us to define a density functional that achieves accurate and reliable prediction of g-tensors, giving confidence in our evaluation of PlAA10 LPMO models. Large models were considered that include all parts of the protein matrix surrounding the Cu site, along with the characteristic second-sphere features of PlAA10. The results uniquely identify the rhombic signal with a five-coordinate Cu ion bearing two water molecules in addition to three N-donor ligands. The axial signal is attributed to a four-coordinate Cu ion where only one of the waters remains bound, as hydroxy. Alternatives that involve decoordination of the histidine brace amino group are unlikely based on energetics and spectroscopy. These results provide a reliable spectroscopy-consistent view on the plasticity of the resting state in PlAA10 LPMO as a foundation for further elucidating structure-property relationships and the formation of catalytically competent species. Our strategy is generally applicable to the study of EPR parameters of mononuclear copper-containing metalloenzymes.

Structural analysis of histone deacetylase 8 mutants associated with Cornelia de Lange Syndrome spectrum disorders.

Cornelia de Lange Syndrome (CdLS) and associated spectrum disorders are characterized by one or more congenital anomalies including distinctive facial features, upper limb abnormalities, intellectual disability, and other symptoms. The molecular genetic basis of CdLS is linked to defects in cohesin, a protein complex that functions in sister chromatid cohesion, chromatin organization, and transcriptional regulation. Histone deacetylase 8 (HDAC8) plays an important role in cohesin function by catalyzing the deacetylation of SMC3, which is required for efficient recycling of the cohesin complex. Missense mutations in HDAC8 have been identified in children diagnosed with CdLS spectrum disorders, and here we outline structure-function relationships for four of these mutations. Specifically, we report the 1.50 Å-resolution structure of the I45T HDAC8-suberoylanilide hydroxamic acid complex, the 1.84 Å-resolution structure of E66D/Y306F HDAC8 complexed with a peptide assay substrate, and the 2.40 Å-resolution structure of G320R HDAC8 complexed with the inhibitor M344. Additionally, we present a computationally generated model of D176G HDAC8. These structures illuminate new structure-function relationships for HDAC8 and highlight the importance of long-range interactions in the protein scaffold that can influence catalytic function.

Understanding the g-tensors of perchlorotriphenylmethyl and Finland-type trityl radicals.

The 285 GHz EPR spectra of perchlorotriphenylmethyl and tetrathiatriarylmethyl radicals in frozen solution have been accurately measured. The relationship between their molecular structures and their g-tensors has been investigated with the aid of DFT calculations, revealing that the degree of spin density delocalization away from the central methylene carbon is an important determining factor of the g-anisotropy. In particular, the small amount of spin densities on the Cl or S heteroatoms at the 2 and 6 positions with respect to the central carbon have the strongest influence. Furthermore, the amount of spin densities on these heteroatoms and thus the anisotropy can be modulated by the protonation (esterification) state of the carboxylate groups at the 4 position. These results provide unique insights into the g-anisotropy of persistent trityl radicals and how it can be tuned.

Phosphorylation of Histone Deacetylase 8: Structural and Mechanistic Analysis of the Phosphomimetic S39E Mutant.

Histone deacetylase (HDAC) enzymes that catalyze removal of acetyl-lysine post-translational modifications are frequently post-translationally modified. HDAC8 is phosphorylated within the deacetylase domain at conserved residue serine 39, which leads to decreased catalytic activity. HDAC8 phosphorylation at S39 is unique in its location and function and may represent a novel mode of deacetylation regulation. To better understand the impact of phosphorylation of HDAC8 on enzyme structure and function, we performed crystallographic, kinetic, and molecular dynamics studies of the S39E HDAC8 phosphomimetic mutant. This mutation decreases the level of deacetylation of peptides derived from acetylated nuclear and cytoplasmic proteins. However, the magnitude of the effect depends on the peptide sequence and the identity of the active site metal ion [Zn(II) vs Fe(II)], with the value of kcat/KM for the mutant decreasing 9- to >200-fold compared to that of wild-type HDAC8. Furthermore, the dissociation rate constant of the active site metal ion increases by ∼10-fold. S39E HDAC8 was crystallized in complex with the inhibitor Droxinostat, revealing that phosphorylation of S39, as mimicked by the glutamate side chain, perturbs local structure through distortion of the L1 loop. Molecular dynamics simulations of both S39E and phosphorylated S39 HDAC8 demonstrate that the perturbation of the L1 loop likely occurs because of the lost hydrogen bond between D29 and S39. Furthermore, the S39 perturbation causes structural changes that propagate through the protein scaffolding to influence function in the active site. These data demonstrate that phosphorylation plays an important regulatory role for HDAC8 by affecting ligand binding, catalytic efficiency, and substrate selectivity.

HDAC8 mutations in Cornelia de Lange syndrome affect the cohesin acetylation cycle.

Cornelia de Lange syndrome (CdLS) is a dominantly inherited congenital malformation disorder, caused by mutations in the cohesin-loading protein NIPBL for nearly 60% of individuals with classical CdLS, and by mutations in the core cohesin components SMC1A (~5%) and SMC3 (<1%) for a smaller fraction of probands. In humans, the multisubunit complex cohesin is made up of SMC1, SMC3, RAD21 and a STAG protein. These form a ring structure that is proposed to encircle sister chromatids to mediate sister chromatid cohesion and also has key roles in gene regulation. SMC3 is acetylated during S-phase to establish cohesiveness of chromatin-loaded cohesin, and in yeast, the class I histone deacetylase Hos1 deacetylates SMC3 during anaphase. Here we identify HDAC8 as the vertebrate SMC3 deacetylase, as well as loss-of-function HDAC8 mutations in six CdLS probands. Loss of HDAC8 activity results in increased SMC3 acetylation and inefficient dissolution of the 'used' cohesin complex released from chromatin in both prophase and anaphase. SMC3 with retained acetylation is loaded onto chromatin, and chromatin immunoprecipitation sequencing analysis demonstrates decreased occupancy of cohesin localization sites that results in a consistent pattern of altered transcription seen in CdLS cell lines with either NIPBL or HDAC8 mutations.

Structural and Functional Influence of the Glycine-Rich Loop G302GGGY on the Catalytic Tyrosine of Histone Deacetylase 8.

Histone deacetylase 8 (HDAC8) catalyzes the hydrolysis of acetyl-l-lysine to yield products l-lysine and acetate through a mechanism in which a nucleophilic water molecule is activated by a histidine general base and a catalytic metal ion (Zn2+ or Fe2+). Acetyl-l-lysine also requires activation by metal coordination and a hydrogen bond with catalytic tyrosine Y306, which also functions in transition state stabilization. Interestingly, Y306 is located in the conserved glycine-rich loop G302GGGY. The potential flexibility afforded by the tetraglycine segment may facilitate induced-fit conformational changes in Y306 between "in" and "out" positions, as observed in related deacetylases. To probe the catalytic importance of the glycine-rich loop in HDAC8, we rigidified this loop by preparing the G302A, G303A, G304A, and G305A mutants and measured their steady state kinetics and determined their X-ray crystal structures. Substantial losses of catalytic efficiency are observed (10-500-fold based on kcat/KM), particularly for G304A HDAC8 and G305A HDAC8. These mutants also exhibit the greatest structural changes for catalytic tyrosine Y306 (1.3-1.7 Å shifts of the phenolic hydroxyl group). Molecular dynamics simulations further indicate that G304 and G305 undergo pronounced structural changes as residue 306 undergoes a transition between "in" and "out" conformations. Thus, the G304A and G305A substitutions likely compromise the position and conformational changes of Y306 required for substrate activation and transition state stabilization. The G302A and G303A substitutions have less severe catalytic consequences, and these substitutions may influence an internal channel through which product acetate is believed to exit.

General Base-General Acid Catalysis in Human Histone Deacetylase 8.

Histone deacetylases (HDACs) regulate cellular processes such as differentiation and apoptosis and are targeted by anticancer therapeutics in development and in the clinic. HDAC8 is a metal-dependent class I HDAC and is proposed to use a general acid-base catalytic pair in the mechanism of amide bond hydrolysis. Here, we report site-directed mutagenesis and enzymological measurements to elucidate the catalytic mechanism of HDAC8. Specifically, we focus on the catalytic function of Y306 and the histidine-aspartate dyads H142-D176 and H143-D183. Additionally, we report X-ray crystal structures of four representative HDAC8 mutants: D176N, D176N/Y306F, D176A/Y306F, and H142A/Y306F. These structures provide a useful framework for understanding enzymological measurements. The pH dependence of kcat/KM for wild-type Co(II)-HDAC8 is bell-shaped with two pKa values of 7.4 and 10.0. The upper pKa reflects the ionization of the metal-bound water molecule and shifts to 9.1 in Zn(II)-HDAC8. The H142A mutant has activity 230-fold lower than that of wild-type HDAC8, but the pKa1 value is not altered. Y306F HDAC8 is 150-fold less active than the wild-type enzyme; crystal structures show that Y306 hydrogen bonds with the zinc-bound substrate carbonyl, poised for transition state stabilization. The H143A and H142A/H143A mutants exhibit activity that is >80000-fold lower than that of wild-type HDAC8; the buried D176N and D176A mutants have significant catalytic effects, with more subtle effects caused by D183N and D183A. These enzymological and structural studies strongly suggest that H143 functions as a single general base-general acid catalyst, while H142 remains positively charged and serves as an electrostatic catalyst for transition state stabilization.

Loss-of-function HDAC8 mutations cause a phenotypic spectrum of Cornelia de Lange syndrome-like features, ocular hypertelorism, large fontanelle and X-linked inheritance.

Cornelia de Lange syndrome (CdLS) is a multisystem genetic disorder with distinct facies, growth failure, intellectual disability, distal limb anomalies, gastrointestinal and neurological disease. Mutations in NIPBL, encoding a cohesin regulatory protein, account for >80% of cases with typical facies. Mutations in the core cohesin complex proteins, encoded by the SMC1A, SMC3 and RAD21 genes, together account for ∼5% of subjects, often with atypical CdLS features. Recently, we identified mutations in the X-linked gene HDAC8 as the cause of a small number of CdLS cases. Here, we report a cohort of 38 individuals with an emerging spectrum of features caused by HDAC8 mutations. For several individuals, the diagnosis of CdLS was not considered prior to genomic testing. Most mutations identified are missense and de novo. Many cases are heterozygous females, each with marked skewing of X-inactivation in peripheral blood DNA. We also identified eight hemizygous males who are more severely affected. The craniofacial appearance caused by HDAC8 mutations overlaps that of typical CdLS but often displays delayed anterior fontanelle closure, ocular hypertelorism, hooding of the eyelids, a broader nose and dental anomalies, which may be useful discriminating features. HDAC8 encodes the lysine deacetylase for the cohesin subunit SMC3 and analysis of the functional consequences of the missense mutations indicates that all cause a loss of enzymatic function. These data demonstrate that loss-of-function mutations in HDAC8 cause a range of overlapping human developmental phenotypes, including a phenotypically distinct subgroup of CdLS.

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 Mycoplana ramosa 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 N(8)-acetylspermidine bearing trifluoromethylketone, thiol, and hydroxamate zinc-binding groups [Decroos, C., Bowman, C. M., and Christianson, D. W. (2013) Bioorg. Med. Chem. 21, 4530], and newly synthesized hydroxamate analogues of shorter, monoacetylated diamines, the most potent of which is the hydroxamate analogue of N-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 N(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.

Variable active site loop conformations accommodate the binding of macrocyclic largazole analogues to HDAC8.

The macrocyclic depsipeptide Largazole is a potent inhibitor of metal-dependent histone deacetylases (HDACs), some of which are drug targets for cancer chemotherapy. Indeed, Largazole partially resembles Romidepsin (FK228), a macrocyclic depsipeptide already approved for clinical use. Each inhibitor contains a pendant side chain thiol that coordinates to the active site Zn(2+) ion, as observed in the X-ray crystal structure of the HDAC8-Largazole complex [Cole, K. E., Dowling, D. P., Boone, M. A., Phillips, A. J., and Christianson, D. W. (2011) J. Am. Chem. Soc. 133, 12474]. Here, we report the X-ray crystal structures of HDAC8 complexed with three synthetic analogues of Largazole in which the depsipeptide ester is replaced with a rigid amide linkage. In two of these analogues, a six-membered pyridine ring is also substituted (with two different orientations) for the five-membered thiazole ring in the macrocycle skeleton. The side chain thiol group of each analogue coordinates to the active site Zn(2+) ion with nearly ideal geometry, thereby preserving the hallmark structural feature of inhibition by Largazole. Surprisingly, in comparison with the binding of Largazole, these analogues trigger alternative conformational changes in loops L1 and L2 flanking the active site. However, despite these structural differences, inhibitory potency is generally comparable to, or just moderately less than, the inhibitory potency of Largazole. Thus, this study reveals important new structure-affinity relationships for the binding of macrocyclic inhibitors to HDAC8.

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(2+)-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 N-(phenylcarbamothioyl)-benzamide, suggesting that HDAC8 activators may serve as possible leads in the therapeutic management of CdLS.

Reactions of amino acids, peptides, and proteins with oxidized metabolites of tris(p-carboxyltetrathiaaryl)methyl radical EPR probes.

Oxidation of the tris(p-carboxyltetrathiaaryl)methyl (TAM) EPR radical probe, TAMa(•), by rat liver microsomes (RLM) + NADPH, or horseradish peroxidase (HRP) + H2O2, or K2IrCl6, led to an intermediate cation, TAMa(+), which was treated with glutathione (GSH), with formation of an adduct, TAMa-SG(•), resulting from the substitution of a TAMa(•) carboxylate group with the SG group. L-α-Amino acids containing a strong nucleophilic residue (NuH), such as L-cysteine or L-histidine, also reacted with TAMa(+), with formation of radical adducts TAMa-Nu(•) in which a carboxylate group of TAMa(•) was replaced with Nu. Other less nucleophilic L-α-amino acids, such as L-arginine, L-serine, L-threonine, L-tyrosine, or L-aspartate, as well as the tetrapeptide H-(Gly)4-OH, reacted with TAMa(+) via their α-NH2 group, with formation of an iminoquinone methide, IQMa, deriving from an oxidative decarboxylation and amination of TAMa(•). Upon reaction of TAMa(+) with L-proline and L-lysine, N-substituted iminoquinone methide adducts, IQMa-Pro and IQMa-Lys, were formed. Finally, preliminary results showed that oxidation of TAMa(•) in the presence of bovine serum albumin (BSA), led to the covalent binding of TAMa-derived metabolites to BSA. Oxidation of another frequently used TAM probe, TAMb(•) (Oxo63), in the presence of GSH, N-acetyl-cysteine methyl ester, or histidine also led to TAMb-Nu(•) adducts equivalent to the corresponding TAMa-Nu(•) adducts, suggesting that the oxidative metabolism of such TAM(•) probes could lead to protein covalent binding. Moreover, the above data describe an easy access to new TAM radical EPR probes coupled to amino acids, peptides or proteins that could be useful for addressing various biological targets.

Repurposing myoglobin into a carbene transferase for a [2,3]-sigmatropic Sommelet-Hauser rearrangement.

New-to-Nature biocatalysis has emerged as a promising tool in organic synthesis thanks to progress in protein engineering. Notably, hemeproteins have been evolved into robust catalysts for carbene and nitrene transfers and related sigmatropic rearrangements. In this work, we report the first example of a [2,3]-sigmatropic Sommelet-Hauser rearrangement initiated by a carbene transfer of the sperm whale myoglobin mutant L29S,H64V,V68F that was previously reported to catalyze the mechanistically similar [2,3]-sigmatropic Doyle-Kirmse rearrangement. This repurposed heme enzyme catalyzes the Sommelet-Hauser rearrangement between ethyl diazoacetate and benzyl thioethers bearing strong electron-withdrawing substituents with good yields and enantiomeric excess. Optimized catalytic conditions in the absence of any reductant led to an increased asymmetric induction with up to 59% enantiomeric excess. This myoglobin mutant is therefore one of the few catalysts for the asymmetric Sommelet-Hauser rearrangement. This work broadens the scope of abiological reactions catalyzed by iron-carbene transferases with a new example of asymmetric sigmatropic rearrangement.

The cohesin ATPase cycle is mediated by specific conformational dynamics and interface plasticity of SMC1A and SMC3 ATPase domains.

Cohesin is key to eukaryotic genome organization and acts throughout the cell cycle in an ATP-dependent manner. The mechanisms underlying cohesin ATPase activity are poorly understood. Here, we characterize distinct steps of the human cohesin ATPase cycle and show that the SMC1A and SMC3 ATPase domains undergo specific but concerted structural rearrangements along this cycle. Specifically, whereas the proximal coiled coil of the SMC1A ATPase domain remains conformationally stable, that of the SMC3 displays an intrinsic flexibility. The ATP-dependent formation of the heterodimeric SMC1A/SMC3 ATPase module (engaged state) favors this flexibility, which is counteracted by NIPBL and DNA binding (clamped state). Opening of the SMC3/RAD21 interface (open-engaged state) stiffens the SMC3 proximal coiled coil, thus constricting together with that of SMC1A the ATPase module DNA-binding chamber. The plasticity of the ATP-dependent interface between the SMC1A and SMC3 ATPase domains enables these structural rearrangements while keeping the ATP gate shut. VIDEO ABSTRACT.

Toward stable electron paramagnetic resonance oximetry probes: synthesis, characterization, and metabolic evaluation of new ester derivatives of a tris-(para-carboxyltetrathiaaryl)methyl (TAM) radical.

Tris(p-carboxyltetrathiaaryl)methyl (TAM) radicals, such as 1a ("Finland" radical), are useful EPR probes for oximetry. However, they are rapidly metabolized by liver microsomes in the presence of NADPH, with the formation of diamagnetic quinone-methide metabolites resulting from an oxidative decarboxylation of one of their carboxylate substituents. In an effort to obtain TAM derivatives potentially more metabolically stable in vivo, we have synthesized four new TAM radicals in which the carboxylate substituents of 1a have been replaced with esters groups bearing various alkyl chains designed to render them water-soluble. The new compounds were completely characterized by UV-vis and EPR spectroscopies, high resolution mass spectrometry (HRMS), and electrochemistry. Two of them were water-soluble enough to undergo detailed microsomal metabolic studies in comparison with 1a. They were found to be stable in the presence of the esterases present in rat liver microsomes and cytosol, and, contrary to 1a, stable to oxidation in the presence of NADPH-supplemented microsomes. A careful study of their possible microsomal reduction under anaerobic or aerobic conditions showed that they were more easily reduced than 1a, in agreement with their higher reduction potentials. They were reduced into the corresponding anions not only under anaerobic conditions but also in the presence of dioxygen. These anions were much more stable than that of 1a and could be characterized by UV-vis spectroscopy, MS, and at the level of their protonated product. However, they were oxidized by O2, giving back to the starting ester radicals and catalyzing a futile cycle of O2 reduction. Such reactions should be considered in the design of future stable EPR probes for oximetry in vivo.

Synthesis and evaluation of N8-acetylspermidine analogues as inhibitors of bacterial acetylpolyamine amidohydrolase.

Polyamines are small essential polycations involved in many biological processes. Enzymes of polyamine metabolism have been extensively studied and are attractive drug targets. Nevertheless, the reversible acetylation of polyamines remains poorly understood. Although eukaryotic N(8)-acetylspermidine deacetylase activity has already been detected and studied, the specific enzyme responsible for this activity has not yet been identified. However, a zinc deacetylase from Mycoplana ramosa, acetylpolyamine amidohydrolase (APAH), has been reported to use various acetylpolyamines as substrates. The recently solved crystal structure of this polyamine deacetylase revealed the formation of an 'L'-shaped active site tunnel at the dimer interface, with ideal dimensions and electrostatic properties for accommodating narrow, flexible, cationic polyamine substrates. Here, we report the design, synthesis, and evaluation of N(8)-acetylspermidine analogues bearing different zinc binding groups as potential inhibitors of APAH. Most of the synthesized compounds exhibit modest potency, with IC50 values in the mid-micromolar range, but compounds bearing hydroxamate or trifluoromethylketone zinc binding groups exhibit enhanced inhibitory potency in the mid-nanomolar range. These inhibitors will enable future explorations of acetylpolyamine function in both prokaryotes and eukaryotes.

First combined in vivo X-ray tomography and high-resolution molecular electron paramagnetic resonance (EPR) imaging of the mouse knee joint taking into account the disappearance kinetics of the EPR probe.

Although laboratory data clearly suggest a role for oxidants (dioxygen and free radicals derived from dioxygen) in the pathogenesis of many age-related and degenerative diseases (such as arthrosis and arthritis), methods to image such species in vivo are still very limited. This methodological problem limits physiopathologic studies about the role of those species in vivo, the effects of their regulation using various drugs, and the evaluation of their levels for diagnosis of degenerative diseases. In vivo electron paramagnetic resonance (EPR) imaging and spectroscopy are unique, noninvasive methods used to specifically detect and quantify paramagnetic species. However, two problems limit their application: the anatomic location of the EPR image in the animal body and the relative instability of the EPR probes. Our aim is to use EPR imaging to obtain physiologic and pathologic information on the mouse knee joint. This article reports the first in vivo EPR image of a small tissue, the mouse knee joint, with good resolution (≈ 160 µm) after intra-articular injection of a triarylmethyl radical EPR probe. It was obtained by combining EPR and x-ray micro-computed tomography for the first time and by taking into account the disappearance kinetics of the EPR probe during image acquisition to reconstruct the image. This multidisciplinary approach opens the way to high-resolution EPR imaging and local metabolism studies of radical species in vivo in different physiologic and pathologic situations.

Oxidative decarboxylation of tris-(p-carboxyltetrathiaaryl)methyl radical EPR probes by peroxidases and related hemeproteins: intermediate formation and characterization of the corresponding cations.

Tris(p-carboxyltetrathiaaryl)methyl radicals (TAM*) are good EPR probes for measurement of dioxygen concentration in biological systems and for EPR imaging. It has been previously reported that these radicals are efficiently oxidized by superoxide, O2*(-), or alkylperoxyl radicals, ROO*, and by liver microsomes via an oxidative decarboxylation mechanism leading to the corresponding quinone-methides (QM). This article shows that peroxidases, such as horseradish peroxidase (HRP), lactoperoxidase (LPO) and prostaglandin synthase (PGHS), and other hemeproteins, such as methemoglobin (metHb), metmyoglobin (metMb) and catalase, also efficiently catalyze the oxidation of TAM* radicals to QM by H2O2 or alkylhydroperoxides. These reactions involve the intermediate formation of the corresponding cations TAM(+) that have also been cleanly generated by K2Ir(IV)Cl6 and characterized by UV-Visible spectroscopy and mass spectrometry, and through their reactions with ascorbate or H2O2. Labelling experiments on HRP-catalyzed oxidation of TAM* to QM using H2(18)O or (18)O2 in the presence of glucose and glucose oxidase (GOX) showed that the oxygen atom incorporated into QM came both from O2 and from H2O. Mechanisms for these reactions in agreement with those data were proposed. Oxidative decarboxylation of TAM* to QM is a new reaction catalyzed by peroxidases. Such reactions should be considered when using TAM* as EPR oximetry probes in vivo or in vitro in complex biological media.

Characterization of a bacterial copper-dependent lytic polysaccharide monooxygenase with an unusual second coordination sphere.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes involved in the degradation of recalcitrant polysaccharides such as cellulose or chitin. LPMOs act in synergy with glycoside hydrolases such as cellulases and chitinases by oxidatively cleaving a number of glycosidic bonds at the surface of their crystalline substrate(s). Besides their role in biomass degradation, some bacterial LPMOs have been found to be virulence factors in some human and insect pathogens. Photorhabdus luminescens is a nematode symbiont bacterium that is pathogenic to a wide range of insects. A single gene encoding a LPMO is found in its genome. In this work, we report the characterization of this LPMO, referred to as PlAA10. Surprisingly, PlAA10 lacks the conserved alanine residue (substituted by an isoleucine) found in the second coordination sphere of the copper-active site in bacterial LPMOs. PlAA10 was found to be catalytically active on both α- and ß-chitin, and exhibits a C1-oxidation regiospecificity, similarly to other chitin-active LPMOs. The 1.6 Å X-ray crystal structure confirmed that PlAA10 adopts the canonical immunoglobulin-like fold typical for LPMOs. The geometry of the copper-active site is not affected by the nearby isoleucine, as also supported by electron paramagnetic resonance. Nevertheless, the bulkier side chain of isoleucine protrudes from the substrate-binding surface. A bioinformatic study on putative bacterial LPMOs unveiled that they exhibit some variability at the conserved active-site alanine position with a substitution in about 15% of all sequences analyzed. DATABASE: Structural data (atomic coordinates and structure factors) reported for PlAA10 are available in the Protein Data Bank under accession number 6T5Z. ENZYMES: PlAA10, EC1.14.99.53.