Unlocking the specificity of antimicrobial peptide interactions for membrane-targeted therapies.

Antimicrobial peptides (AMPs) are increasingly recognized as potent therapeutic agents, with their selective affinity for pathological membranes, low toxicity profile, and minimal resistance development making them particularly attractive in the pharmaceutical landscape. This study offers a comprehensive analysis of the interaction between specific AMPs, including magainin-2, pleurocidin, CM15, LL37, and clavanin, with lipid bilayer models of very different compositions that have been ordinarily used as biological membrane models of healthy mammal, cancerous, and bacterial cells. Employing unbiased molecular dynamics simulations and metadynamics techniques, we have deciphered the intricate mechanisms by which these peptides recognize pathogenic and pathologic lipid patterns and integrate into lipid assemblies. Our findings reveal that the transverse component of the peptide's hydrophobic dipole moment is critical for membrane interaction, decisively influencing the molecule's orientation and expected therapeutic efficacy. Our approach also provides insight on the kinetic and dynamic dependence on the peptide orientation in the axial and azimuthal angles when coming close to the membrane. The aim is to establish a robust framework for the rational design of peptide-based, membrane-targeted therapies, as well as effective quantitative descriptors that can facilitate the automated design of novel AMPs for these therapies using machine learning methods.

ß-l-Arabinofurano-cyclitol Aziridines Are Covalent Broad-Spectrum Inhibitors and Activity-Based Probes for Retaining ß-l-Arabinofuranosidases.

GH127 and GH146 microorganismal retaining ß-l-arabinofuranosidases, expressed by human gut microbiomes, feature an atypical catalytic domain and an unusual mechanism of action. We recently reported that both Bacteroides thetaiotaomicron BtGH146 and Bifidobacterium longum HypBA1 are inhibited by ß-l-arabinofuranosyl cyclophellitol epoxide, supporting the action of a zinc-coordinated cysteine as a catalytic nucleophile, where in most retaining GH families, an aspartate or glutamate is employed. This work presents a panel of ß-l-arabinofuranosyl cyclophellitol epoxides and aziridines as mechanism-based BtGH146/HypBA1 inhibitors and activity-based probes. The ß-l-arabinofuranosyl cyclophellitol aziridines both inhibit and label ß-l-arabinofuranosidase efficiently (however with different activities), whereas the epoxide-derived probes favor BtGH146 over HypBA1. These findings are accompanied by X-ray structural analysis of the unmodified ß-l-arabinofuranosyl cyclophellitol aziridine in complex with both isozymes, which were shown to react by nucleophilic opening of the aziridine, at the pseudoanomeric carbon, by the active site cysteine nucleophile to form a stable thioether bond. Altogether, our activity-based probes may serve as chemical tools for the detection and identification of low-abundance ß-l-arabinofuranosidases in complex biological samples.

In silico modelling of the function of disease-related CAZymes.

In silico modelling of proteins comprises a diversity of computational tools aimed to obtain structural, electronic, and/or dynamic information about these biomolecules, capturing mechanistic details that are challenging to experimental approaches, such as elusive enzyme-substrate complexes, short-lived intermediates, and reaction transition states (TS). The present article gives the reader insight on the use of in silico modelling techniques to understand complex catalytic reaction mechanisms of carbohydrate-active enzymes (CAZymes), along with the underlying theory and concepts that are important in this field. We start by introducing the significance of carbohydrates in nature and the enzymes that process them, CAZymes, highlighting the conformational flexibility of their carbohydrate substrates. Three commonly used in silico methods (classical molecular dynamics (MD), hybrid quantum mechanics/molecular mechanics (QM/MM), and enhanced sampling techniques) are described for nonexpert readers. Finally, we provide three examples of the application of these methods to unravel the catalytic mechanisms of three disease-related CAZymes: ß-galactocerebrosidase (GALC), responsible for Krabbe disease; α-mannoside ß-1,6-N-acetylglucosaminyltransferase V (MGAT5), involved in cancer; and O-fucosyltransferase 1 (POFUT1), involved in several human diseases such as leukemia and the Dowling-Degos disease.

4-O-Substituted Glucuronic Cyclophellitols are Selective Mechanism-Based Heparanase Inhibitors.

Degradation of the extracellular matrix (ECM) supports tissue integrity and homeostasis, but is also a key factor in cancer metastasis. Heparanase (HPSE) is a mammalian ECM-remodeling enzyme with ß-D-endo-glucuronidase activity overexpressed in several malignancies, and is thought to facilitate tumor growth and metastasis. By this virtue, HPSE is considered an attractive target for the development of cancer therapies, yet to date no HPSE inhibitors have progressed to the clinic. Here we report on the discovery of glucurono-configured cyclitol derivatives featuring simple substituents at the 4-O-position as irreversible HPSE inhibitors. We show that these compounds, unlike glucurono-cyclophellitol, are selective for HPSE over ß-D-exo-glucuronidase (GUSB), also in platelet lysate. The observed selectivity is induced by steric and electrostatic interactions of the substituents at the 4-O-position. Crystallographic analysis supports this rationale for HPSE selectivity, and computer simulations provide insights in the conformational preferences and binding poses of the inhibitors, which we believe are good starting points for the future development of HPSE-targeting antimetastatic cancer drugs.

Cysteine Nucleophiles in Glycosidase Catalysis: Application of a Covalent ß-l-Arabinofuranosidase Inhibitor.

The recent discovery of zinc-dependent retaining glycoside hydrolases (GHs), with active sites built around a Zn(Cys)3 (Glu) coordination complex, has presented unresolved mechanistic questions. In particular, the proposed mechanism, depending on a Zn-coordinated cysteine nucleophile and passing through a thioglycosyl enzyme intermediate, remains controversial. This is primarily due to the expected stability of the intermediate C-S bond. To facilitate the study of this atypical mechanism, we report the synthesis of a cyclophellitol-derived ß-l-arabinofuranosidase inhibitor, hypothesised to react with the catalytic nucleophile to form a non-hydrolysable adduct analogous to the mechanistic covalent intermediate. This ß-l-arabinofuranosidase inhibitor reacts exclusively with the proposed cysteine thiol catalytic nucleophiles of representatives of GH families 127 and 146. X-ray crystal structures determined for the resulting adducts enable MD and QM/MM simulations, which provide insight into the mechanism of thioglycosyl enzyme intermediate breakdown. Leveraging the unique chemistry of cyclophellitol derivatives, the structures and simulations presented here support the assignment of a zinc-coordinated cysteine as the catalytic nucleophile and illuminate the finely tuned energetics of this remarkable metalloenzyme clan.

Structural and kinetic features of aldehyde dehydrogenase 1A (ALDH1A) subfamily members, cancer stem cell markers active in retinoic acid biosynthesis.

Aldehyde dehydrogenases catalyze the NAD(P)+-dependent oxidation of aldehydes to their corresponding carboxylic acids. The three-dimensional structures of the human ALDH1A enzymes were recently obtained, while a complete kinetic characterization of them, under the same experimental conditions, is lacking. We show that the three enzymes, ALDH1A1, ALDH1A2 and ALDH1A3, have similar topologies, although with decreasing volumes in their substrate-binding pockets. The activity with aliphatic and retinoid aldehydes was characterized side-by-side, using an improved HPLC-based method for retinaldehyde. Hexanal was the most efficient substrate. ALDH1A1 displayed lower Km values with hexanal, trans-2-hexenal and citral, compared to ALDH1A2 and ALDH1A3. ALDH1A2 was the best enzyme for the lipid peroxidation product, 4-hydroxy-2-nonenal, in terms of kcat/Km. The catalytic efficiency towards all-trans and 9-cis-retinaldehyde was in general lower than for alkanals and alkenals. ALDH1A2 and ALDH1A3 showed higher catalytic efficiency for all-trans-retinaldehyde. The lower specificity of ALDH1A3 for 9-cis-retinaldehyde against the all-trans- isomer might be related to the smaller volume of its substrate-binding pocket. Magnesium inhibited ALDH1A1 and ALDH1A2, while it activated ALDH1A3, which is consistent with cofactor dissociation being the rate-limiting step for ALDH1A1 and ALDH1A2, and deacylation for ALDH1A3, with hexanal as a substrate. We mutated both ALDH1A1 (L114P) and ALDH1A2 (N475G, A476V, L477V, N478S) to mimic their counterpart substrate-binding pockets. ALDH1A1 specificity for citral was traced to residue 114 and to residues 458 to 461. Regarding retinaldehyde, the mutants did not show significant differences with their respective wild-type forms, suggesting that the mutated residues are not critical for retinoid specificity.

Modeling catalytic reaction mechanisms in glycoside hydrolases.

Modeling catalysis in carbohydrate-active enzymes is a daunting challenge because of the high flexibility and diversity of both enzymes and carbohydrates. Glycoside hydrolases (GHs) are an illustrative example, where conformational changes and subtle interactions have been shown to be critical for catalysis. GHs have pivotal roles in industry (e.g. biofuel or detergent production) and biomedicine (e.g. targets for cancer and diabetes), and thus, a huge effort is devoted to unveil their molecular mechanisms. Besides experimental techniques, computational methods have served to provide an in-depth understanding of GH mechanisms, capturing complex reaction coordinates and the conformational itineraries that substrates follow during the whole catalytic pathway, providing a framework that ultimately may assist the engineering of these enzymes and the design of new inhibitors.

Structural and Mechanistic Insights into the Catalytic-Domain-Mediated Short-Range Glycosylation Preferences of GalNAc-T4.

Mucin-type O-glycosylation is initiated by a family of polypeptide GalNAc-transferases (GalNAc-Ts) which are type-II transmembrane proteins that contain Golgi luminal catalytic and lectin domains that are connected by a flexible linker. Several GalNAc-Ts, including GalNAc-T4, show both long-range and short-range prior glycosylation specificity, governed by their lectin and catalytic domains, respectively. While the mechanism of the lectin-domain-dependent glycosylation is well-known, the molecular basis for the catalytic-domain-dependent glycosylation of glycopeptides is unclear. Herein, we report the crystal structure of GalNAc-T4 bound to the diglycopeptide GAT*GAGAGAGT*TPGPG (containing two α-GalNAc glycosylated Thr (T*), the PXP motif and a "naked" Thr acceptor site) that describes its catalytic domain glycopeptide GalNAc binding site. Kinetic studies of wild-type and GalNAc binding site mutant enzymes show the lectin domain GalNAc binding activity dominates over the catalytic domain GalNAc binding activity and that these activities can be independently eliminated. Surprisingly, a flexible loop protruding from the lectin domain was found essential for the optimal activity of the catalytic domain. This work provides the first structural basis for the short-range glycosylation preferences of a GalNAc-T.

Redesigning the Coumarin Scaffold into Small Bright Fluorophores with Far-Red to Near-Infrared Emission and Large Stokes Shifts Useful for Cell Imaging.

Among the palette of previously described fluorescent organic molecules, coumarins are ideal candidates for developing cellular and molecular imaging tools due to their high cell permeability and minimal perturbation of living systems. However, blue-to-cyan fluorescence emission is usually difficult in in vivo applications due to the inherent toxicity and poor tissue penetration of short visible light wavelengths. Here, we introduce a new family of coumarin-based fluorophores, nicknamed COUPY, with promising photophysical properties, including emission in the far-red/near-infrared (NIR) region, large Stokes shifts, high photostability, and excellent brightness. COUPY fluorophores were efficiently synthesized in only three linear synthetic steps from commercially available precursors, with the N-alkylation of a pyridine moiety being the key step at the end of the synthetic route, as it allows for the tuning of the photophysical properties of the resulting dye. Owing to their low molecular weights, COUPY dyes show excellent cell permeability and accumulate selectively in nucleoli and/or mitochondria of HeLa cells, as their far-red/NIR fluorescence emission is easily detected at a concentration as low as 0.5 µM after an incubation of only 20 min. We anticipate that these coumarin scaffolds will open a way to the development of novel coumarin-based far-red to NIR emitting fluorophores with potential applications for organelle imaging and biomolecule labeling.

Selective Derivatization of N-Terminal Cysteines Using Cyclopentenediones.

The outcome of the Michael-type reaction between thiols and 2,2-disubstituted cyclopentenediones varies depending on the thiol. Stable compounds with two fused rings were formed upon reaction with 1,2-aminothiols (such as N-terminal cysteines in peptides). Other thiols gave reversibly Michael-type adducts that were in equilibrium with the starting materials. This differential reactivity allows differently placed cysteines to be distinguished and has been exploited to prepare bioconjugates incorporating two or three different moieties.

Have We Improved Pain Control in Cancer Patients? A Multicenter Study of Ambulatory and Hospitalized Cancer Patients.

BACKGROUND: Pain in cancer patients is recognized as a major health problem, yet few studies of both inpatient and outpatient populations have been carried out. OBJECTIVE: The study objective was to assess the frequency, type, and characteristics of pain in adult cancer patients, including both inpatients and outpatients. METHODS: This cross-sectional study involved 1064 adult cancer patients (437 outpatients and 627 inpatients) from 44 hospitals and/or long-term-care centers in Catalonia, Spain. Cancer patients suffering from pain of any etiology for ≥2 weeks and/or under analgesic treatment ≥2 weeks were enrolled. Demographic and pain data were collected. The Spanish version of the Brief Pain Inventory was used to assess pain. RESULTS: Pain frequency was 55.3%. Pain was less frequent in outpatients than inpatients (41.6% versus 64.7%; p<0.001), although median pain duration was longer in outpatients (20 versus 6 weeks; p<0.001). Pain was assessable in 333 patients, and intensity was similar in both out- and inpatients; however, outpatients reported less improvement, less pain interference with daily life, and less pain related to the cancer per se. In both groups, patients with multiple myeloma (73%), breast (65%), and lung cancer (61%) were most likely to report pain. CONCLUSIONS: Pain in cancer patients, both ambulatory and hospitalized, remains a challenge for health care professionals, health administrators, and stakeholders. Our study reveals the high level of pain and distress that cancer patients continue to suffer, a problem that is particularly notable in outpatients due to the intensity and duration of the pain.

Dynamic interplay between catalytic and lectin domains of GalNAc-transferases modulates protein O-glycosylation.

Protein O-glycosylation is controlled by polypeptide GalNAc-transferases (GalNAc-Ts) that uniquely feature both a catalytic and lectin domain. The underlying molecular basis of how the lectin domains of GalNAc-Ts contribute to glycopeptide specificity and catalysis remains unclear. Here we present the first crystal structures of complexes of GalNAc-T2 with glycopeptides that together with enhanced sampling molecular dynamics simulations demonstrate a cooperative mechanism by which the lectin domain enables free acceptor sites binding of glycopeptides into the catalytic domain. Atomic force microscopy and small-angle X-ray scattering experiments further reveal a dynamic conformational landscape of GalNAc-T2 and a prominent role of compact structures that are both required for efficient catalysis. Our model indicates that the activity profile of GalNAc-T2 is dictated by conformational heterogeneity and relies on a flexible linker located between the catalytic and the lectin domains. Our results also shed light on how GalNAc-Ts generate dense decoration of proteins with O-glycans.

Substrate-guided front-face reaction revealed by combined structural snapshots and metadynamics for the polypeptide N-acetylgalactosaminyltransferase†2.

The retaining glycosyltransferase GalNAc-T2 is a member of a large family of human polypeptide GalNAc-transferases that is responsible for the post-translational modification of many cell-surface proteins. By the use of combined structural and computational approaches, we provide the first set of structural snapshots of the enzyme during the catalytic cycle and combine these with quantum-mechanics/molecular-mechanics (QM/MM) metadynamics to unravel the catalytic mechanism of this retaining enzyme at the atomic-electronic level of detail. Our study provides a detailed structural rationale for an ordered bi-bi kinetic mechanism and reveals critical aspects of substrate recognition, which dictate the specificity for acceptor Thr versus Ser residues and enforce a front-face SN i-type reaction in which the substrate N-acetyl sugar substituent coordinates efficient glycosyl transfer.

Retinaldehyde is a substrate for human aldo-keto reductases of the 1C subfamily.

Human AKR (aldo-keto reductase) 1C proteins (AKR1C1-AKR1C4) exhibit relevant activity with steroids, regulating hormone signalling at the pre-receptor level. In the present study, investigate the activity of the four human AKR1C enzymes with retinol and retinaldehyde. All of the enzymes except AKR1C2 showed retinaldehyde reductase activity with low Km values (~1 µM). The kcat values were also low (0.18-0.6 min-1), except for AKR1C3 reduction of 9-cis-retinaldehyde whose kcat was remarkably higher (13 min-1). Structural modelling of the AKR1C complexes with 9-cis-retinaldehyde indicated a distinct conformation of Trp227, caused by changes in residue 226 that may contribute to the activity differences observed. This was partially supported by the kinetics of the AKR1C3 R226P mutant. Retinol/retinaldehyde conversion, combined with the use of the inhibitor flufenamic acid, indicated a relevant role for endogenous AKR1Cs in retinaldehyde reduction in MCF-7 breast cancer cells. Overexpression of AKR1C proteins depleted RA (retinoic acid) transactivation in HeLa cells treated with retinol. Thus AKR1Cs may decrease RA levels in vivo. Finally, by using lithocholic acid as an AKR1C3 inhibitor and UVI2024 as an RA receptor antagonist, we provide evidence that the pro-proliferative action of AKR1C3 in HL-60 cells involves the RA signalling pathway and that this is in part due to the retinaldehyde reductase activity of AKR1C3.

Human and rodent aldo-keto reductases from the AKR1B subfamily and their specificity with retinaldehyde.

NADP(H)-dependent cytosolic aldo-keto reductases (AKR) are mostly monomeric enzymes which fold into a typical (α/ß)(8)-barrel structure. Substrate specificity and inhibitor selectivity are determined by interaction with residues located in three highly variable loops (A, B, and C). Based on sequence identity, AKR have been grouped into families, namely AKR1-AKR15, containing multiple subfamilies. Two human enzymes from the AKR1B subfamily (AKR1B1 and AKR1B10) are of special interest. AKR1B1 (aldose reductase) is related to secondary diabetic complications, while AKR1B10 is induced in cancer cells and is highly active with all-trans-retinaldehyde. Residues interacting with all-trans-retinaldehyde and differing between AKR1B1 and AKR1B10 are Leu125Lys and Val131Ala (loop A), Leu301Val, Ser303Gln, and Cys304Ser (loop C). Recently, we demonstrated the importance of Lys125 as a determinant of AKR1B10 specificity for retinoids. Residues 301 and 304 are also involved in interactions with substrates or inhibitors, and thus we checked their contribution to retinoid specificity. We also extended our study with retinoids to rodent members of the AKR1B subfamily: AKR1B3 (aldose reductase), AKR1B7 (mouse vas deferens protein), AKR1B8 (fibroblast-growth factor 1-regulated protein), and AKR1B9 (Chinese hamster ovary reductase), which were tested against all-trans isomers of retinaldehyde and retinol. All enzymes were active with retinaldehyde, but with k(cat) values (0.02-0.52 min(-1)) much lower than that of AKR1B10 (27 min(-1)). None of the enzymes showed oxidizing activity with retinol. Since these enzymes (except AKR1B3) have Lys125, other residues should account for retinaldehyde specificity. Here, by using site-directed mutagenesis and molecular modeling, we further delineate the contribution of residues 301 and 304. We demonstrate that besides Lys125, Ser304 is a major structural determinant for all-trans-retinaldehyde specificity of AKR1B10.

A neutral zwitterionic molecular solid.

We report on the acid ethylenedithiotetrathiafulvaleneamidoglycine (EDT-TTF-CO-NH-CH(2)-CO(2)H; 1; EDT-TTF=ethylenedithiotetrathiafulvalene) and the 1:1 adduct [(EDT-TTF)(·+)-CO-NH-CH(2)-(CO(2))(-)][(EDT-TTF)-CO-NH-CH(2)-(CO(2)H)]·CH(3)OH (2), a new type of hydrogen-bonded, 1:1 acid/zwitterion hybrid embrace of redox peptidics into a two-dimensional architecture, an example of a system deliberately fashioned so that oxidation of π-conjugated cores toward the radical-cation form would interfere with the activity of the appended ionizable residues in the presence of a templating base during crystal growth. First-principles calculations demonstrate that, notwithstanding preconceived ideas, a metallic state is more stable than the hole-localized alternatives for a neat 1:1 neutral acid/zwitterion hybrid. The inhomogeneous Coulomb field associated with proton-shared, interstacks O-H···O hydrogen bonds between the ionizable residues distributed on both sides of the two-dimensional π-conjugated framework leads, however, to a weak hole localization responsible for the activated but high conductivity of 1 S cm(-1). This situation is reminiscent of the role of the environment on electron transfer in tetraheme cytochrome c, in which the protonation state of a heme propionate becomes paramount, or ion-gated transport phenomena in biology. These observations open rather intriguing opportunities for the construction of electronic systems at the interface of chemistry and biology.

Modulation of Abeta42 fibrillogenesis by glycosaminoglycan structure.

The role of amyloid ß (Aß) peptide in the onset and progression of Alzheimer's disease is linked to the presence of soluble Aß species. Sulfated glycosaminoglycans (GAGs) promote Aß fibrillogenesis and reduce the toxicity of the peptide in neuronal cell cultures, but a satisfactory rationale to explain these effects at the molecular level has not been provided yet. We have used circular dichroism, Fourier transform infrared spectroscopy, fluorescence microscopy and spectroscopy, protease digestion, atomic force microscopy (AFM), and molecular dynamics simulations to characterize the association of the 42-residue fragment Aß(42) with sulfated GAGs, hyaluronan, chitosan, and poly(vinyl sulfate) (PVS). Our results indicate that the formation of stable Aß(42) fibrils is promoted by polymeric GAGs with negative charges placed in-frame with the 4.8-Å separating Aß(42) monomers within protofibrillar ß-sheets. Incubation of Aß(42) with excess sulfated GAGs and hyaluronan increased amyloid fibril content and resistance to proteolysis 2- to 5-fold, whereas in the presence of the cationic polysaccharide chitosan, Aß(42) fibrillar species were reduced by 25% and sensitivity to protease degradation increased ∼3-fold. Fibrils of intermediate stability were obtained in the presence of PVS, an anionic polymer with more tightly packed charges than GAGs. Important structural differences between Aß(42) fibrils induced by PVS and Aß(42) fibrils obtained in the presence of GAGs and hyaluronan were observed by AFM, whereas mainly precursor protofibrillar forms were detected after incubation with chitosan. Computed binding energies per peptide from -11.2 to -13.5 kcal/mol were calculated for GAGs and PVS, whereas a significantly lower value of -7.4 kcal/mol was obtained for chitosan. Taken together, our data suggest a simple and straightforward mechanism to explain the role of GAGs as enhancers of the formation of insoluble Aß(42) fibrils trapping soluble toxic forms.

Molecular mechanism of the glycosylation step catalyzed by Golgi alpha-mannosidase II: a QM/MM metadynamics investigation.

Golgi alpha-mannosidase II (GMII), a member of glycoside hydrolase family 38, cleaves two mannosyl residues from GlcNAcMan(5)GlcNAc(2) as part of the N-linked glycosylation pathway. To elucidate the molecular and electronic details of the reaction mechanism, in particular the conformation of the substrate at the transition state, we performed quantum mechanics/molecular mechanics metadynamics simulations of the glycosylation reaction catalyzed by GMII. The calculated free energy of activation for mannosyl glycosylation (23 kcal/mol) agrees very well with experiments, as does the conformation of the glycon mannosyl ring in the product of the glycosylation reaction (the covalent intermediate). In addition, we provide insight into the electronic aspects of the molecular mechanism that were not previously available. We show that the substrate adopts an (O)S(2)/B(2,5) conformation in the GMII Michaelis complex and that the nucleophilic attack occurs before complete departure of the leaving group, consistent with a D(N)A(N) reaction mechanism. The transition state has a clear oxacarbenium ion (OCI) character, with the glycosylation reaction following an (O)S(2)/B(2,5) --> B(2,5) [TS] --> (1)S(5) itinerary, agreeing with an earlier proposal based on comparing alpha- and beta-mannanases. The simulations also demonstrate that an active-site Zn ion helps to lengthen the O2'-H(O2') bond when the substrate acquires OCI character, relieving the electron deficiency of the OCI-like species. Our results can be used to explain the potency of recently formulated GMII anticancer inhibitors, and they are potentially relevant in deriving new inhibitors.

Analysis of the reaction coordinate of alpha-L-fucosidases: a combined structural and quantum mechanical approach.

The enzymatic hydrolysis of alpha-L-fucosides is of importance in cancer, bacterial infections, and fucosidosis, a neurodegenerative lysosomal storage disorder. Here we show a series of snapshots along the reaction coordinate of a glycoside hydrolase family GH29 alpha-L-fucosidase unveiling a Michaelis (ES) complex in a (1)C(4) (chair) conformation and a covalent glycosyl-enzyme intermediate in (3)S(1) (skew-boat). First principles metadynamics simulations on isolated alpha-L-fucose strongly support a (1)C(4)<-->(3)H(4)<-->(3)S(1) conformational itinerary for the glycosylation step of the reaction mechanism and indicate a strong "preactivation" of the (1)C(4) complex to nucleophilic attack as reflected by free energy, C1-O1/O5-C1 bond length elongation/reduction, C1-O1 bond orientation, and positive charge development around the anomeric carbon. Analysis of an imino sugar inhibitor is consistent with tight binding of a chair-conformed charged species.

Essential role of proximal histidine-asparagine interaction in mammalian peroxidases.

In heme enzymes belonging to the peroxidase-cyclooxygenase superfamily the proximal histidine is in close interaction with a fully conserved asparagine. The crystal structure of a mixture of glycoforms of myeloperoxidase (MPO) purified from granules of human leukocytes prompted us to revise the orientation of this asparagine and the protonation status of the proximal histidine. The data we present contrast with previous MPO structures, but are strongly supported by molecular dynamics simulations. Moreover, comprehensive analysis of published lactoperoxidase structures suggest that the described proximal heme architecture is a general structural feature of animal heme peroxidases. Its importance is underlined by the fact that the MPO variant N421D, recombinantly expressed in mammalian cell lines, exhibited modified spectral properties and diminished catalytic activity compared with wild-type recombinant MPO. It completely lost its ability to oxidize chloride to hypochlorous acid, which is a characteristic feature of MPO and essential for its role in host defense. The presented crystal structure of MPO revealed further important differences compared with the published structures including the extent of glycosylation, interaction between light and heavy polypeptides, as well as heme to protein covalent bonds. These data are discussed with respect to biosynthesis and post-translational maturation of MPO as well as to its peculiar biochemical and biophysical properties.